DNA polymerases with improved activity

ABSTRACT

Disclosed are DNA polymerases having increased reverse transcriptase efficiency relative to a corresponding, unmodified polymerase. The polymerases are useful in a variety of disclosed primer extension methods. Also disclosed are related compositions, including recombinant nucleic acids, vectors, and host cells, which are useful, e.g., for production of the DNA polymerases.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application claims benefit of priority to U.S. ProvisionalPatent Application No. 61/568,375, filed Dec. 8, 2011, which isincorporated herein by reference in its entirety.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file -140-1.TXT, created on Dec. 5,2012, 131,072 bytes, machine format IBM-PC, MS-Windows operating system,is hereby incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

The present invention provides DNA polymerases with improved activities,including increased reverse transcriptase efficiency, mismatchtolerance, extension rate and/or tolerance of reverse transcriptase (RT)and polymerase inhibitors, as well as use of such polymerases in variousapplications, including nucleic acid polynucleotide extension andamplification.

BACKGROUND OF THE INVENTION

DNA polymerases are responsible for the replication and maintenance ofthe genome, a role that is central to accurately transmitting geneticinformation from generation to generation. DNA polymerases function incells as the enzymes responsible for the synthesis of DNA. Theypolymerize deoxyribonucleoside triphosphates in the presence of a metalactivator, such as Mg²⁺, in an order dictated by the DNA template orpolynucleotide template that is copied. In vivo, DNA polymerasesparticipate in a spectrum of DNA synthetic processes including DNAreplication, DNA repair, recombination, and gene amplification. Duringeach DNA synthetic process, the DNA template is copied once or at most afew times to produce identical replicas. In contrast, in vitro, DNAreplication can be repeated many times such as, for example, duringpolymerase chain reaction (see, e.g., U.S. Pat. No. 4,683,202 toMullis).

In the initial studies with polymerase chain reaction (PCR), the DNApolymerase was added at the start of each round of DNA replication (seeU.S. Pat. No. 4,683,202, supra). Subsequently, it was determined thatthermostable DNA polymerases could be obtained from bacteria that growat elevated temperatures, and that these enzymes need to be added onlyonce (see U.S. Pat. No. 4,889,818 to Gelfand and U.S. Pat. No. 4,965,188to Mullis). At the elevated temperatures used during PCR, these enzymesare not irreversibly inactivated. As a result, one can carry outrepetitive cycles of polymerase chain reactions without adding freshenzymes at the start of each synthetic addition process. DNApolymerases, particularly thermostable polymerases, are the key to alarge number of techniques in recombinant DNA studies and in medicaldiagnosis of disease. For diagnostic applications in particular, atarget nucleic acid sequence may be only a small portion of the DNA orRNA in question, so it may be difficult to detect the presence of atarget nucleic acid sequence without amplification.

The overall folding pattern of DNA polymerases resembles the human righthand and contains three distinct subdomains of palm, fingers, and thumb.(See Beese et al., Science 260:352-355, 1993); Patel et al.,Biochemistry 34:5351-5363, 1995). While the structure of the fingers andthumb subdomains vary greatly between polymerases that differ in sizeand in cellular functions, the catalytic palm subdomains are allsuperimposable. For example, motif A, which interacts with the incomingdNTP and stabilizes the transition state during chemical catalysis, issuperimposable with a mean deviation of about one Å amongst mammalianpol α and prokaryotic pol I family DNA polymerases (Wang et al., Cell89:1087-1099, 1997). Motif A begins structurally at an antiparallelβ-strand containing predominantly hydrophobic residues and continues toan α-helix. The primary amino acid sequence of DNA polymerase activesites is exceptionally conserved. In the case of motif A, for example,the sequence DYSQIELR (SEQ ID NO:22) is retained in polymerases fromorganisms separated by many millions years of evolution, including,e.g., Thermus aquaticus, Chlamydia trachomatis, and Escherichia coli.

In addition to being well-conserved, the active site of DNA polymeraseshas also been shown to be relatively mutable, capable of accommodatingcertain amino acid substitutions without reducing DNA polymeraseactivity significantly. (See, e.g., U.S. Pat. No. 6,602,695 to Patel etal.). Such mutant DNA polymerases can offer various selective advantagesin, e.g., diagnostic and research applications comprising nucleic acidsynthesis reactions.

There are at least two steps in the enzymatic process of DNApolymerization; 1) the incorporation of the incoming nucleotide and 2)the extension of the newly incorporated nucleotide. The overallfaithfulness or “fidelity” of the DNA polymerase is generally thought ofas a conglomerate of these two enzymatic activities, but the steps aredistinct. A DNA polymerase may misincorporate the incoming nucleotide,but if it is not efficiently extended the extension rate will beseverely decreased and overall product formation would be minimal.Alternatively, it is possible to have a DNA polymerase misincorporatethe incoming nucleotide and readily misextend the newly formed mismatch.In this case, the overall extension rate would be high, but the overallfidelity would be low. An example of this type of enzyme would be ES112DNA polymerase (E683R Z05 DNA polymerase; see U.S. Pat. No. 7,179,590,entitled “High temperature reverse transcription using mutant DNApolymerases” filed Mar. 30, 2001 by Smith et al., which is incorporatedby reference) when using Mn²⁺ as the divalent metal ion activator. Theenzyme has a very high efficiency because unlike typical DNA polymerasesthat tend to hesitate/stall when a mismatch is encountered, the ES112DNA polymerase readily extends the mismatch. The phenotype displayed inES112 is more pronounced during the RT step, presumably because ofstructural effects of the RNA/DNA heteroduplex vs. the DNA/DNAhomoduplex. A second example would be if the DNA polymerase does notreadily misincorporate (may be even less likely to misincorporate), butdoes have increased capacity to misextend a mismatch. In this case, thefidelity is not significantly altered for the overall product. Ingeneral, this type of enzyme is more favorable for extension reactionsthan the characteristics of ES112 in Mn²⁺ because the fidelity of theproduct is improved. However this attribute can be utilized to allow themisextension of a mismatched oligonucleotide primer such as when anoligonucleotide primer of a single sequence is hybridized to a targetthat has sequence heterogeneity (e.g., viral targets), but the normal orlower misincorporation rate allows for completion of DNA synthesisbeyond the original oligonucleotide primer. An example of this type ofDNA polymerase is Z05 D580G DNA polymerase. (see U.S. Patent PublicationNo. 2009/0148891 entitled “DNA Polymerases and Related Methods” filedOct. 17, 2007 by Bauer et. al., which is incorporated by reference).This type of activity is referred to as “mismatch tolerant” because itis more tolerant to mismatches in the oligonucleotide primer. While theexamples above have discussed primer extension type reactions, theactivity can be more significant in reactions such as RT-PCR and PCRwhere primer extension is reoccurring frequently. Data suggests thatwhile enzymes such as Z05 D580G are more “tolerant” to mismatches, theyalso have enhanced ability to extend oligonucleotide primers containingmodified bases (eg., t-butyl benzyl modified bases) or in the presenceof DNA binding dyes such as SYBR Green I (see U.S. Patent PublicationNo. 2009/028053 entitled “Improved DNA Polymerases and Related Methods”filed Apr. 16, 2009 by Bauer et al., which is incorporated byreference).

Reverse transcription polymerase chain reaction (RT-PCR) is a techniqueused in many applications to detect/and or quantify RNA targets byamplification. In order to amplify RNA targets by PCR, it is necessaryto first reverse transcribe the RNA template into cDNA. Typically,RT-PCR assays rely on a non-thermostable reverse transcriptase (RNAdependent DNA polymerase), derived from a mesophilic organism, for theinitial cDNA synthesis step (RT). An additional thermostable DNApolymerase is required for amplification of cDNA to tolerate elevatedtemperatures required for nucleic acid denaturation in PCR. There areseveral potential benefits of using thermoactive or thermostable DNApolymerases engineered to perform more efficient reverse transcriptionfor RT-PCR assays. Increased reverse transcriptase activity coupled withthe ability to use higher reverse transcription incubation temperatures,that allow for relaxing of RNA template secondary structure, can resultin overall higher cDNA synthesis efficiency and assay sensitivity.Higher temperature incubation could also increase specificity byreducing false priming in the reverse transcription step. Enzymes withimproved reverse transcription efficiency can simplify assay design byallowing for reduced RT incubation times and/or enzyme concentration.When using dUTP and UNG, nonspecific extension products containing dUMPthat are formed during nonstringent set-up conditions are degraded byUNG and cannot be utilized either as primers or as templates. When usinga non-thermostable reverse transcriptase (RNA dependent DNA polymerase)derived from a mesophilic organism, it is not possible to utilize thedUTP and UNG methodologies. (Myers, T. W. et al., Amplification of RNA:High Temperature Reverse Transcription and DNA Amplification withThermus thermophilus DNA Polymerase, in PCR Strategies, Innis, M. A.,Gelfand, D. H., and Sninsky, J. J., Eds., Academic Press, San Diego,Calif., 58-68, (1995)). However, the use of a thermoactive orthermostable DNA polymerase of the invention for the reversetranscription step enables the reaction to be completely compatible withthe utilization of the dUTP/uracil N-glycosylase (UNG) carry-overprevention system (Longo et al., Use of Uracil DNA Glycosylase toControl Carry-over Contamination in Polymerase Chain Reactions. Gene93:125-128, (1990). In addition to providing carry-over contaminationcontrol, the use of dUTP and UNG provides a “hot-start” to reducenonspecific amplification (Innis and Gelfand 1999).

BRIEF SUMMARY OF THE INVENTION

Provided herein are DNA polymerases having improved activities,including increased reverse transcriptase efficiency, mismatchtolerance, extension rate and/or tolerance of RT and polymeraseinhibitors, relative to a corresponding, unmodified control polymerase,and methods of making and using such DNA polymerases. In someembodiments, the improved DNA polymerase has increased reversetranscriptase efficiency as compared with a control DNA polymerase. Insome embodiments, the improved DNA polymerase has the same orsubstantially similar DNA-dependent polymerase activity as compared witha control DNA polymerase. Thus, in some embodiments, the improved DNApolymerase comprises an amino acid sequence that is substantiallyidentical (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or95% identical) to SEQ ID NO:1, wherein the amino acid of the DNApolymerase corresponding to position 640 of SEQ ID NO:1 is any aminoacid other than 1 or V. In some embodiments, the control DNA polymerasehas the same amino acid sequence as the DNA polymerase except that theamino acid of the control DNA polymerase corresponding to position 640of SEQ ID NO:1 is I or V. For example, in some embodiments, the aminoacid at the position corresponding to position 640 of SEQ ID NO:1 of theimproved polymerase is selected from G, A, R, F, W, P, S, T, C, Y, N, Q,D, E, K, L, M, or H. In some embodiments, the amino acid at the positioncorresponding to position 640 of SEQ ID NO:1 of the improved polymeraseis F.

In some embodiments, the improved DNA polymerase further comprises anamino acid sequence that is substantially identical (e.g., at leastabout 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical) to SEQ IDNO:1, wherein the amino acid of the DNA polymerase corresponding toposition 580 of SEQ ID NO:1 is any amino acid other than D or E. In someembodiments, the amino acid of the DNA polymerase corresponding toposition 580 of SEQ ID NO:1 is any amino acid other than D. In someembodiments, the amino acid of the DNA polymerase corresponding toposition 580 of SEQ ID NO:1 is selected from the group consisting of L,G, T, Q, A, S, N, R, and K. In some embodiments, the amino acid of theDNA polymerase corresponding to position 580 of SEQ ID NO:1 is G.

In some embodiments, the improved DNA polymerase further comprises anamino acid sequence that is substantially identical (e.g., at leastabout 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical) to SEQ IDNO:1, wherein the amino acid of the DNA polymerase corresponding toposition 709 of SEQ ID NO:1 is any amino acid other than I. In someembodiments, the amino acid of the DNA polymerase corresponding toposition 709 of SEQ ID NO:1 is selected from the group consisting of K,R, S, G, and A. In some embodiments, the amino acid of the DNApolymerase corresponding to position 709 of SEQ ID NO:1 is K.

In some embodiments, the improved DNA polymerase comprises an amino acidsequence that is substantially identical (e.g., at least about 60%, 65%,70%, 75%, 80%, 85%, 90%, or 95% identical) to SEQ ID NO:1, wherein theamino acid of the DNA polymerase corresponding to position 640 of SEQ IDNO:1 is any amino acid other than I, the amino acid corresponding toposition 580 of SEQ ID NO:1 is any amino acid other than D or E, and theamino acid corresponding to position 709 of SEQ ID NO:1 is any aminoacid other than I. Thus, in some embodiments, the improved DNApolymerase comprises an amino acid sequence that is substantiallyidentical (e.g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or95% identical) to SEQ ID NO:1, wherein the amino acid of the DNApolymerase corresponding to position 640 of SEQ ID NO:1 is F, the aminoacid corresponding to position 580 of SEQ ID NO:1 is G, and the aminoacid corresponding to position 709 of SEQ ID NO:1 is K.

In some embodiments, the improved DNA polymerase has increased reversetranscriptase efficiency without a substantial decrease in DNA-dependentpolymerase activity compared with a control DNA polymerase, wherein theamino acid of the DNA polymerase corresponding to position 640 of SEQ IDNO:1 is any amino acid other than I, and the amino acid corresponding toposition 709 of SEQ ID NO:1 is any amino acid other than I, and whereinthe control DNA polymerase has the same amino acid sequence as the DNApolymerase except that the amino acid of the control DNA polymerasecorresponding to position 640 of SEQ ID NO:1 is I and the amino acidcorresponding to position 709 of SEQ ID NO:1 is I. Thus, in someembodiments, the amino acid of the DNA polymerase corresponding toposition 640 of SEQ ID NO:1 is F, and the amino acid corresponding toposition 709 of SEQ ID NO:1 is K. In some embodiments, the improved DNApolymerase further comprises an amino acid substitution at the aminoacid corresponding to position 580 of SEQ ID NO:1. Thus, in someembodiments, the amino acid of the DNA polymerase corresponding toposition 640 of SEQ ID NO:1 is any amino acid other than I, the aminoacid corresponding to position 709 of SEQ ID NO:1 is any amino acidother than I, and the amino acid corresponding to position 580 of SEQ IDNO:1 is any amino acid other than D or E. In some embodiments, the aminoacid of the DNA polymerase corresponding to position 640 of SEQ ID NO:1is F, the amino acid corresponding to position 709 of SEQ ID NO:1 is K,and the amino acid corresponding to position 580 of SEQ ID NO:1 is G.

Various DNA polymerases are amenable to mutation according to thepresent invention. Particularly suitable are thermostable polymerases,including wild-type or naturally occurring thermostable polymerases fromvarious species of thermophilic bacteria, as well as syntheticthermostable polymerases derived from such wild-type or naturallyoccurring enzymes by amino acid substitution, insertion, or deletion, orother modification. Exemplary unmodified forms of polymerase include,e.g., CS5, CS6 or Z05 DNA polymerase, or a functional DNA polymerasehaving at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequenceidentity thereto. Other unmodified polymerases include, e.g., DNApolymerases from any of the following species of thermophilic bacteria(or a functional DNA polymerase having at least 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% amino acid sequence identity to such a polymerase): Thermotogamaritima; Thermus aquaticus; Thermus thermophilus; Thermus flavus;Thermus filiformis; Thermus sp. sps17; Thermus sp. Z05; Thermotoganeopolitana; Thermosipho africanus; Thermus caldophilus, Deinococcusradiodurans, Bacillus stearothermophilus or Bacillus caldotenax.Suitable polymerases also include those having reverse transcriptase(RT) activity and/or the ability to incorporate unconventionalnucleotides, such as ribonucleotides or other 2′-modified nucleotides.

While thermostable DNA polymerases possessing efficient reversetranscription activity are particularly suited for performing RT-PCR,especially single enzyme RT-PCR, thermoactive, but not thermostable DNApolymerases possessing efficient reverse transcription activity also areamenable to mutation according to the present invention. For example,the attributes of increased reverse transcriptase efficiency, mismatchtolerance, extension rate, and/or tolerance of RT inhibitors are usefulfor the RT step in an RT-PCR and this step does not need to be performedat temperatures that would inactivate a thermoactive but notthermostable DNA polymerase. Following the RT step, a thermostable DNApolymerase could either be added or it could already be included in thereaction mixture to perform the PCR amplification step. For example, theimproved DNA polymerase described herein can be combined with a secondthermostable DNA polymerase prior to the RT step in a buffer suitablefor extension and amplification of RNA and DNA templates, as describedin the Examples. Examples of suitable thermostable DNA polymerases aredescribed in U.S. Pat. No. 4,889,818 to Gelfand et al., and U.S. Pat.Nos. 5,773,258 and 5,677,152 to Birch et al., which are expresslyincorporated by reference herein in their entirety. In some embodiments,the second thermostable DNA polymerase is AmpliTaq® DNA polymerase(Deoxy-nucleoside triphosphate: DNA Deoxynucleotidyltransferase,E.C.2.7.7.7). In some embodiments, the second thermostable DNApolymerase is a reversibly inactivated thermostable polymerase, asdescribed below. In one embodiment, the reversibly inactivatedthermostable polymerase is AmpliTaq Gold® DNA polymerase (Roche AppliedScience, Indianapolis, Ind., USA). This second methodology wouldespecially benefit by using a chemically modified thermostable DNApolymerase (or other HotStart technology to inactivate the thermostableDNA polymerase) so that it would not be fully active during the RT step.An example of a thermoactive but not thermostable DNA polymerasepossessing efficient reverse transcription activity is the DNApolymerase from Carboxydothermus hydrogenoformans (Chy; SEQ ID NO:39).See, e.g., U.S. Pat. Nos. 6,468,775 and 6,399,320.

In some embodiments, the DNA polymerase has at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% amino acid sequence identity to a polymerase selected from thegroup consisting of:

-   -   (a) a Thermus sp. Z05 DNA polymerase (Z05) (SEQ ID NO:1);    -   (b) a Thermus aquaticus DNA polymerase (Taq) (SEQ ID NO:2);    -   (c) a Thermus filiformis DNA polymerase (Tfi) (SEQ ID NO:3);    -   (d) a Thermus flavus DNA polymerase (Tfl) (SEQ ID NO:4);    -   (e) a Thermus sp. sps17 DNA polymerase (Sps17) (SEQ ID NO:5);    -   (f) a Thermus thermophilus DNA polymerase (Tth) (SEQ ID NO:6);        and    -   (g) a Thermus caldophilus DNA polymerase (Tca) (SEQ ID NO:7)    -   (h) Carboxydothermus hydrogenoformans DNA polymerase (Chy) (SEQ        ID NO:39)

In some embodiments, the DNA polymerase is a Thermotoga DNA polymerase.For example, in some embodiments, the DNA polymerase has at least 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% amino acid sequence identity to a polymeraseselected from the group consisting of:

-   -   (a) a Thermotoga maritima DNA polymerase (Tma) (SEQ ID NO:34);    -   (b) a Thermotoga neopolitana DNA polymerase (Tne) (SEQ ID        NO:35);

In some embodiments, the DNA polymerase has at least 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% amino acid sequence identity to SEQ ID NO:1. In someembodiments, the DNA polymerase is a Thermus sp. Z05 DNA polymerase(Z05) DNA polymerase (i.e., SEQ ID NO:1), and the amino acid at position640 is any amino acid other than I. For example, in some embodiments,the amino acid at position 640 is selected from G, A, V, R, F, W, P, S,T, C, Y, N, Q, D, E, K, L, M or H. In some embodiments, the DNApolymerase is a Z05 DNA polymerase, and the amino acid at position 640is F. In some embodiments, the DNA polymerase is a Z05 DNA polymerasefurther comprising a substitution at position 580, and the amino acid atposition 580 is any amino acid other than D or E. In some embodiments,the DNA polymerase is a Z05 DNA polymerase, and the amino acid atposition 580 is any amino acid other than D. In some embodiments, theDNA polymerase is a Z05 DNA polymerase, and the amino acid at position580 is selected from the group consisting of L, G, T, Q, A, S, N, R, andK. In some embodiments, the DNA polymerase is a Z05 DNA polymerase, andthe amino acid at position 580 is G. In some embodiments, the DNApolymerase is a Z05 DNA polymerase further comprising a substitution atposition 709, and the amino acid at position 709 is any amino acid otherthan I. In some embodiments, the DNA polymerase is a Z05 DNA polymerase,and the amino acid at position 709 is selected from the group consistingof K, R, S, G, and A. In some embodiments, the DNA polymerase is a Z05DNA polymerase, and the amino acid at position 709 is K.

In some embodiments, the control DNA polymerase is a Z05, Z05 D580G, orZ05 D580G I709K polymerase.

The mutant or improved polymerases can include other, non-substitutionalmodifications. One such modification is a thermally reversible covalentmodification that inactivates the enzyme, but which is reversed toactivate the enzyme upon incubation at an elevated temperature, such asa temperature typically used for polynucleotide extension. Exemplaryreagents for such thermally reversible modifications are described inU.S. Pat. Nos. 5,773,258 and 5,677,152 to Birch et al., which areexpressly incorporated by reference herein in their entirety.

In some embodiments, the reverse transcriptase activity is determined byperforming real-time RT-PCR amplification and detection of a Hepatitis CVirus (HCV) transcript generated from the first 800 bases of HCVgenotype Ib 5′NTR in pSP64 poly(A) (Promega). Two or more reactionmixtures can have titrated numbers of copies of the Hepatitis C Virus(HCV) transcript (e.g., 1:5 titrations, 1:10 titrations, e.g., 10,000copies, 1000 copies, 100 copies, 10 copies, 1 copy, 0 copies in severalreaction mixtures). The reverse transcriptase ability of a polymerase ofthe invention can be compared to the reverse transcriptase ability of areference polymerase (e.g., a naturally occurring, unmodified, orcontrol polymerase), over a preselected unit of time, as describedherein. Polymerases with improved reverse transcriptase ability willamplify the transcript with greater efficiency, or will require a lowernumber of PCR cycles to amplify the transcript (i.e., exhibit a lower Cpvalue, as calculated herein), in comparison to a naturally occurring orunmodified polymerase. Moreover, in some embodiments, polymerases withimproved RT function also have improved replication of long RNA (e.g.,at least 500 or 1000 or 2000 or 5000 or more nucleotides long)templates. In some embodiments, the improved reverse transcriptaseefficiency includes a shorter reverse transcription time in comparisonto a control polymerase. Thus, in some embodiments, polymerases withincreased reverse transcriptase efficiency will reverse transcribe anRNA template faster than a control or reference polymerase.

In various other aspects, the present invention provides a recombinantnucleic acid encoding a mutant or improved DNA polymerase as describedherein, a vector comprising the recombinant nucleic acid, and a hostcell transformed with the vector. In certain embodiments, the vector isan expression vector. Host cells comprising such expression vectors areuseful in methods of the invention for producing the mutant or improvedpolymerase by culturing the host cells under conditions suitable forexpression of the recombinant nucleic acid. The polymerases of theinvention may be contained in reaction mixtures and/or kits. Theembodiments of the recombinant nucleic acids, host cells, vectors,expression vectors, reaction mixtures and kits are as described aboveand herein.

In yet another aspect, a method for conducting polynucleotide extensionis provided. The method generally includes contacting a DNA polymerasehaving increased reverse transcriptase efficiency, mismatch tolerance,extension rate and/or tolerance of RT and polymerase inhibitors asdescribed herein with a primer, a polynucleotide template, andnucleoside triphosphates under conditions suitable for extension of theprimer, thereby producing an extended primer. The polynucleotidetemplate can be, for example, an RNA or DNA template. The nucleotidetriphosphates can include unconventional nucleotides such as, e.g.,ribonucleotides and/or labeled nucleotides. Further, the primer and/ortemplate can include one or more nucleotide analogs. In some variations,the polynucleotide extension method is a method for polynucleotideamplification that includes contacting the mutant or improved DNApolymerase with a primer pair, the polynucleotide template, and thenucleoside triphosphates under conditions suitable for amplification ofthe polynucleotide. The polynucleotide extension reaction can be, e.g.,PCR, isothermal extension, or sequencing (e.g., 454 sequencingreaction). The polynucleotide template can be from any type ofbiological sample.

Optionally, the primer extension reaction comprises an actual orpotential inhibitor of a reference or unmodified polymerase. Theinhibitor can inhibit the nucleic acid extension rate and/or the reversetranscription efficiency of a reference or unmodified (control)polymerase. In some embodiments, the inhibitor is hemoglobin, or adegradation product thereof. For example, in some embodiments, thehemoglobin degradation product is a heme breakdown product, such ashemin, hematoporphyrin, or bilirubin. In some embodiments, the inhibitoris an iron-chelator or a purple pigment. In other embodiments, theinhibitor is heparin or melanin. In certain embodiments, the inhibitoris an intercalating dye. In some embodiments, the intercalating dye is[2-[N-bis-(3-dimethylaminopropyl)-amino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium]+.In some embodiments, the intercalating dye is[2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium]+.In some embodiments, the intercalating dye is not[2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium]+.In some embodiments, the conditions suitable for extension compriseMg⁺⁺. In some embodiments, the conditions suitable for extensioncomprise Mn⁺⁺.

The present invention also provides a kit useful in such apolynucleotide extension method. Generally, the kit includes at leastone container providing a mutant or improved DNA polymerase as describedherein. In certain embodiments, the kit further includes one or moreadditional containers providing one or more additional reagents. Forexample, in specific variations, the one or more additional containersprovide nucleoside triphosphates; a buffer suitable for polynucleotideextension; and/or one or more primer or probe polynucleotides,hybridizable, under polynucleotide extension conditions, to apredetermined polynucleotide template. The polynucleotide template canbe from any type of biological sample.

Further provided are reaction mixtures comprising the polymerases of theinvention. The reaction mixtures can also contain a template nucleicacid (DNA and/or RNA), one or more primer or probe polynucleotides,nucleoside triphosphates (including, e.g., deoxyribonucleosidetriphosphates, ribonucleoside triphosphates, labeled nucleosidetriphosphates, unconventional nucleoside triphosphates), buffers, salts,labels (e.g., fluorophores). In some embodiments, the reaction mixturescomprise an iron chelator or a purple dye. In certain embodiments, thereaction mixtures comprise hemoglobin, or a degradation product ofhemoglobin. For example, in certain embodiments, the degradationproducts of hemoglobin include heme breakdown products such as hemin,hematin, hematophoryn, and bilirubin. In other embodiments, the reactionmixtures comprise heparin or a salt thereof. Optionally, the reactionmixture comprises an intercalating dye (including but not limited tothose described above or elsewhere herein). In certain embodiments, thereaction mixture contains a template nucleic acid that is isolated fromblood. In other embodiments, the template nucleic acid is RNA and thereaction mixture comprises heparin or a salt thereof.

In some embodiments, the reaction mixture comprises two or morepolymerases. For example, in some embodiments, the reaction mixturecomprises an improved DNA polymerase having increased reversetranscription efficiency (e.g., increased activity extending anRNA-template) as described herein, and another polymerase havingDNA-dependent polymerase activity. In one embodiment, the reactionmixture comprises a blend of an improved DNA polymerase having increasedreverse transcription efficiency as described herein, and a secondthermostable DNA-dependent polymerase. The second thermostableDNA-dependent polymerase can be a reversibly modified polymerase asdescribed above such that the enzyme is inactive at temperaturessuitable for the reverse transcription step, but is activated undersuitable conditions, for example, at elevated temperatures of about 90°C. to 100° C. for a period of time up to about 12 minutes. Suitableconditions for activation of a reversibly inactivated thermostablepolymerase are provided, for example, in a Hot Start PCR reaction, asdescribed in the Examples. Examples of suitable second thermostableDNA-dependent polymerases are described in U.S. Pat. Nos. 5,773,258 and5,677,152 to Birch et al., supra.

Further embodiments of the invention are described herein.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although essentially anymethods and materials similar to those described herein can be used inthe practice or testing of the present invention, only exemplary methodsand materials are described. For purposes of the present invention, thefollowing terms are defined below.

The terms “a,” “an,” and “the” include plural referents, unless thecontext clearly indicates otherwise.

An “amino acid” refers to any monomer unit that can be incorporated intoa peptide, polypeptide, or protein. As used herein, the term “aminoacid” includes the following twenty natural or genetically encodedalpha-amino acids: alanine (Ala or A), arginine (Arg or R), asparagine(Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine(Gln or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K),methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P),serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine(Tyr or Y), and valine (Val or V). In cases where “X” residues areundefined, these should be defined as “any amino acid.” The structuresof these twenty natural amino acids are shown in, e.g., Stryer et al.,Biochemistry, 5^(th) ed., Freeman and Company (2002), which isincorporated by reference. Additional amino acids, such asselenocysteine and pyrrolysine, can also be genetically coded for(Stadtman (1996) “Selenocysteine,” Annu Rev Biochem. 65:83-100 and Ibbaet al. (2002) “Genetic code: introducing pyrrolysine,” Curr Biol.12(13):R464-R466, which are both incorporated by reference). The term“amino acid” also includes unnatural amino acids, modified amino acids(e.g., having modified side chains and/or backbones), and amino acidanalogs. See, e.g., Zhang et al. (2004) “Selective incorporation of5-hydroxytryptophan into proteins in mammalian cells,” Proc. Natl. Acad.Sci. U.S.A. 101(24):8882-8887, Anderson et al. (2004) “An expandedgenetic code with a functional quadruplet codon” Proc. Natl. Acad. Sci.U.S.A. 101(20):7566-7571, Ikeda et al. (2003) “Synthesis of a novelhistidine analogue and its efficient incorporation into a protein invivo,” Protein Eng. Des. Sel. 16(9):699-706, Chin et al. (2003) “AnExpanded Eukaryotic Genetic Code,” Science 301(5635):964-967, James etal. (2001) “Kinetic characterization of ribonuclease S mutantscontaining photoisomerizable phenylazophenylalanine residues,” ProteinEng. Des. Sel. 14(12):983-991, Kohrer et al. (2001) “Import of amber andochre suppressor tRNAs into mammalian cells: A general approach tosite-specific insertion of amino acid analogues into proteins,” Proc.Natl. Acad. Sci. U.S.A. 98(25):14310-14315, Bacher et al. (2001)“Selection and Characterization of Escherichia coli Variants Capable ofGrowth on an Otherwise Toxic Tryptophan Analogue,” J. Bacteriol.183(18):5414-5425, Hamano-Takaku et al. (2000) “A Mutant Escherichiacoli Tyrosyl-tRNA Synthetase Utilizes the Unnatural Amino AcidAzatyrosine More Efficiently than Tyrosine,” J. Biol. Chem.275(51):40324-40328, and Budisa et al. (2001) “Proteins with{beta}-(thienopyrrolyl)alanines as alternative chromophores andpharmaceutically active amino acids,” Protein Sci. 10(7):1281-1292,which are each incorporated by reference.

To further illustrate, an amino acid is typically an organic acid thatincludes a substituted or unsubstituted amino group, a substituted orunsubstituted carboxy group, and one or more side chains or groups, oranalogs of any of these groups. Exemplary side chains include, e.g.,thiol, seleno, sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxyl,hydrazine, cyano, halo, hydrazide, alkenyl, alkynl, ether, borate,boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine,aldehyde, ester, thioacid, hydroxylamine, or any combination of thesegroups. Other representative amino acids include, but are not limitedto, amino acids comprising photoactivatable cross-linkers, metal bindingamino acids, spin-labeled amino acids, fluorescent amino acids,metal-containing amino acids, amino acids with novel functional groups,amino acids that covalently or noncovalently interact with othermolecules, photocaged and/or photoisomerizable amino acids, radioactiveamino acids, amino acids comprising biotin or a biotin analog,glycosylated amino acids, other carbohydrate modified amino acids, aminoacids comprising polyethylene glycol or polyether, heavy atomsubstituted amino acids, chemically cleavable and/or photocleavableamino acids, carbon-linked sugar-containing amino acids, redox-activeamino acids, amino thioacid containing amino acids, and amino acidscomprising one or more toxic moieties.

The term “biological sample” encompasses a variety of sample typesobtained from an organism and can be used in a diagnostic or monitoringassay. The term encompasses urine, urine sediment, blood, saliva, andother liquid samples of biological origin, solid tissue samples, such asa biopsy specimen or tissue cultures or cells derived therefrom and theprogeny thereof. The term encompasses samples that have been manipulatedin any way after their procurement, such as by treatment with reagents,solubilization, sedimentation, or enrichment for certain components. Theterm encompasses a clinical sample, and also includes cells in cellculture, cell supernatants, cell lysates, serum, plasma, biologicalfluids, and tissue samples.

The term “mutant,” in the context of DNA polymerases of the presentinvention, means a polypeptide, typically recombinant, that comprisesone or more amino acid substitutions relative to a corresponding,functional DNA polymerase.

The term “unmodified form,” in the context of a mutant polymerase, is aterm used herein for purposes of defining a mutant DNA polymerase of thepresent invention: the term “unmodified form” refers to a functional DNApolymerase that has the amino acid sequence of the mutant polymeraseexcept at one or more amino acid position(s) specified as characterizingthe mutant polymerase. Thus, reference to a mutant DNA polymerase interms of (a) its unmodified form and (b) one or more specified aminoacid substitutions means that, with the exception of the specified aminoacid substitution(s), the mutant polymerase otherwise has an amino acidsequence identical to the unmodified form in the specified motif. The“unmodified polymerase” (and therefore also the modified form havingincreased reverse transcriptase efficiency, mismatch tolerance,extension rate and/or tolerance of RT and polymerase inhibitors) maycontain additional mutations to provide desired functionality, e.g.,improved incorporation of dideoxyribonucleotides, ribonucleotides,ribonucleotide analogs, dye-labeled nucleotides, modulating 5′-nucleaseactivity, modulating 3′-nuclease (or proofreading) activity, or thelike. Accordingly, in carrying out the present invention as describedherein, the unmodified form of a DNA polymerase is predetermined. Theunmodified form of a DNA polymerase can be, for example, a wild-typeand/or a naturally occurring DNA polymerase, or a DNA polymerase thathas already been intentionally modified. An unmodified form of thepolymerase is preferably a thermostable DNA polymerase, such as DNApolymerases from various thermophilic bacteria, as well as functionalvariants thereof having substantial sequence identity to a wild-type ornaturally occurring thermostable polymerase. Such variants can include,for example, chimeric DNA polymerases such as, for example, the chimericDNA polymerases described in U.S. Pat. Nos. 6,228,628 and 7,148,049,which are incorporated by reference herein in their entirety. In certainembodiments, the unmodified form of a polymerase has reversetranscriptase (RT) activity.

The term “thermostable polymerase,” refers to an enzyme that is stableto heat, is heat resistant, and retains sufficient activity to effectsubsequent polynucleotide extension reactions and does not becomeirreversibly denatured (inactivated) when subjected to the elevatedtemperatures for the time necessary to effect denaturation ofdouble-stranded nucleic acids. The heating conditions necessary fornucleic acid denaturation are well known in the art and are exemplifiedin, e.g., U.S. Pat. Nos. 4,683,202, 4,683,195, and 4,965,188, which areincorporated herein by reference. As used herein, a thermostablepolymerase is suitable for use in a temperature cycling reaction such asthe polymerase chain reaction (“PCR”). Irreversible denaturation forpurposes herein refers to permanent and complete loss of enzymaticactivity. For a thermostable polymerase, enzymatic activity refers tothe catalysis of the combination of the nucleotides in the proper mannerto form polynucleotide extension products that are complementary to atemplate nucleic acid strand. Thermostable DNA polymerases fromthermophilic bacteria include, e.g., DNA polymerases from Thermotogamaritima, Thermus aquaticus, Thermus thermophilus, Thermus flavus,Thermus filiformis, Thermus species sps17, Thermus species Z05, Thermuscaldophilus, Bacillus caldotenax, Thermotoga neopolitana, andThermosipho africanus.

The term “thermoactive” refers to an enzyme that maintains catalyticproperties at temperatures commonly used for reverse transcription oranneal/extension steps in RT-PCR and/or PCR reactions (i.e., 45-80° C.).Thermostable enzymes are those which are not irreversibly inactivated ordenatured when subjected to elevated temperatures necessary for nucleicacid denaturation. Thermoactive enzymes may or may not be thermostable.Thermoactive DNA polymerases can be DNA or RNA dependent fromthermophilic species or from mesophilic species including, but notlimited to, Escherichia coli, Moloney murine leukemia viruses, and Avianmyoblastosis virus.

As used herein, a “chimeric” protein refers to a protein whose aminoacid sequence represents a fusion product of subsequences of the aminoacid sequences from at least two distinct proteins. A chimeric proteintypically is not produced by direct manipulation of amino acidsequences, but, rather, is expressed from a “chimeric” gene that encodesthe chimeric amino acid sequence. In certain embodiments, for example,an unmodified form of a mutant DNA polymerase of the present inventionis a chimeric protein that consists of an amino-terminal (N-terminal)region derived from a Thermus species DNA polymerase and acarboxy-terminal (C-terminal) region derived from Tma DNA polymerase.The N-terminal region refers to a region extending from the N-terminus(amino acid position 1) to an internal amino acid. Similarly, theC-terminal region refers to a region extending from an internal aminoacid to the C-terminus.

The term “aptamer” refers to a single-stranded DNA that recognizes andbinds to DNA polymerase, and efficiently inhibits the polymeraseactivity as described in U.S. Pat. No. 5,693,502, hereby expresslyincorporated by reference herein in its entirety. Use of aptamer anddUTP/UNG in RT-PCR is also discussed, for example, in Smith, E. S. etal, (Amplification of RNA: High-temperature Reverse Transcription andDNA Amplification with a Magnesium-activated Thermostable DNAPolymerase, in PCR Primer: A Laboratory Manual, 2nd Edition,Dieffenbach, C. W. and Dveksler, G. S., Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 211-219, (2003)).

In the context of mutant DNA polymerases, “correspondence” to anothersequence (e.g., regions, fragments, nucleotide or amino acid positions,or the like) is based on the convention of numbering according tonucleotide or amino acid position number and then aligning the sequencesin a manner that maximizes the percentage of sequence identity. An aminoacid “corresponding to position [α] of [specific sequence]” refers to anamino acid in a polypeptide of interest that aligns with the equivalentamino acid of a specified sequence. Generally, as described herein, theamino acid corresponding to a position of a polymerase can be determinedusing an alignment algorithm such as BLAST as described below. Becausenot all positions within a given “corresponding region” need beidentical, non-matching positions within a corresponding region may beregarded as “corresponding positions.” Accordingly, as used herein,referral to an “amino acid position corresponding to amino acid position[α]” of a specified DNA polymerase refers to equivalent positions, basedon alignment, in other DNA polymerases and structural homologues andfamilies. In some embodiments of the present invention, “correspondence”of amino acid positions are determined with respect to a region of thepolymerase comprising one or more motifs of SEQ ID NO:1, 2, 3, 4, 5, 6,7, 32, 33, 34, 35, 36, 37, or 39. When a polymerase polypeptide sequencediffers from SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 32, 33, 34, 35, 36, 37, or39 (e.g., by changes in amino acids or addition or deletion of aminoacids), it may be that a particular mutation associated with improvedactivity as discussed herein will not be in the same position number asit is in SEQ ID NOS:1, 2, 3, 4, 5, 6, 7, 32, 33, 34, 35, 36, 37, or 39.This is illustrated, for example, in Table 1.

“Recombinant,” as used herein, refers to an amino acid sequence or anucleotide sequence that has been intentionally modified by recombinantmethods. By the term “recombinant nucleic acid” herein is meant anucleic acid, originally formed in vitro, in general, by themanipulation of a nucleic acid by restriction endonucleases, in a formnot normally found in nature. Thus an isolated, mutant DNA polymerasenucleic acid, in a linear form, or an expression vector formed in vitroby ligating DNA molecules that are not normally joined, are bothconsidered recombinant for the purposes of this invention. It isunderstood that once a recombinant nucleic acid is made and reintroducedinto a host cell, it will replicate non-recombinantly, i.e., using thein vivo cellular machinery of the host cell rather than in vitromanipulations; however, such nucleic acids, once produced recombinantly,although subsequently replicated non-recombinantly, are still consideredrecombinant for the purposes of the invention. A “recombinant protein”is a protein made using recombinant techniques, i.e., through theexpression of a recombinant nucleic acid as depicted above.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, a promoteror enhancer is operably linked to a coding sequence if it affects thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to facilitatetranslation.

The term “host cell” refers to both single-cellular prokaryote andeukaryote organisms (e.g., bacteria, yeast, and actinomycetes) andsingle cells from higher order plants or animals when being grown incell culture.

The term “vector” refers to a piece of DNA, typically double-stranded,which may have inserted into it a piece of foreign DNA. The vector ormay be, for example, of plasmid origin. Vectors contain “replicon”polynucleotide sequences that facilitate the autonomous replication ofthe vector in a host cell. Foreign DNA is defined as heterologous DNA,which is DNA not naturally found in the host cell, which, for example,replicates the vector molecule, encodes a selectable or screenablemarker, or encodes a transgene. The vector is used to transport theforeign or heterologous DNA into a suitable host cell. Once in the hostcell, the vector can replicate independently of or coincidental with thehost chromosomal DNA, and several copies of the vector and its insertedDNA can be generated. In addition, the vector can also contain thenecessary elements that permit transcription of the inserted DNA into anmRNA molecule or otherwise cause replication of the inserted DNA intomultiple copies of RNA. Some expression vectors additionally containsequence elements adjacent to the inserted DNA that increase thehalf-life of the expressed mRNA and/or allow translation of the mRNAinto a protein molecule. Many molecules of mRNA and polypeptide encodedby the inserted DNA can thus be rapidly synthesized.

The term “nucleotide,” in addition to referring to the naturallyoccurring ribonucleotide or deoxyribonucleotide monomers, shall hereinbe understood to refer to related structural variants thereof, includingderivatives and analogs, that are functionally equivalent with respectto the particular context in which the nucleotide is being used (e.g.,hybridization to a complementary base), unless the context clearlyindicates otherwise.

The term “nucleic acid” or “polynucleotide” refers to a polymer that canbe corresponded to a ribose nucleic acid (RNA) or deoxyribose nucleicacid (DNA) polymer, or an analog thereof. This includes polymers ofnucleotides such as RNA and DNA, as well as synthetic forms, modified(e.g., chemically or biochemically modified) forms thereof, and mixedpolymers (e.g., including both RNA and DNA subunits). Exemplarymodifications include methylation, substitution of one or more of thenaturally occurring nucleotides with an analog, internucleotidemodifications such as uncharged linkages (e.g., methyl phosphonates,phosphotriesters, phosphoamidates, carbamates, and the like), pendentmoieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen,and the like), chelators, alkylators, and modified linkages (e.g., alphaanomeric nucleic acids and the like). Also included are syntheticmolecules that mimic polynucleotides in their ability to bind to adesignated sequence via hydrogen bonding and other chemicalinteractions. Typically, the nucleotide monomers are linked viaphosphodiester bonds, although synthetic forms of nucleic acids cancomprise other linkages (e.g., peptide nucleic acids as described inNielsen et al. (Science 254:1497-1500, 1991). A nucleic acid can be orcan include, e.g., a chromosome or chromosomal segment, a vector (e.g.,an expression vector), an expression cassette, a naked DNA or RNApolymer, the product of a polymerase chain reaction (PCR), anoligonucleotide, a probe, and a primer. A nucleic acid can be, e.g.,single-stranded, double-stranded, or triple-stranded and is not limitedto any particular length. Unless otherwise indicated, a particularnucleic acid sequence optionally comprises or encodes complementarysequences, in addition to any sequence explicitly indicated.

The term “oligonucleotide” refers to a nucleic acid that includes atleast two nucleic acid monomer units (e.g., nucleotides). Anoligonucleotide typically includes from about six to about 175 nucleicacid monomer units, more typically from about eight to about 100 nucleicacid monomer units, and still more typically from about 10 to about 50nucleic acid monomer units (e.g., about 15, about 20, about 25, about30, about 35, or more nucleic acid monomer units). The exact size of anoligonucleotide will depend on many factors, including the ultimatefunction or use of the oligonucleotide. Oligonucleotides are optionallyprepared by any suitable method, including, but not limited to,isolation of an existing or natural sequence, DNA replication oramplification, reverse transcription, cloning and restriction digestionof appropriate sequences, or direct chemical synthesis by a method suchas the phosphotriester method of Narang et al. (Meth. Enzymol. 68:90-99,1979); the phosphodiester method of Brown et al. (Meth. Enzymol.68:109-151, 1979); the diethylphosphoramidite method of Beaucage et al.(Tetrahedron Lett. 22:1859-1862, 1981); the triester method of Matteucciet al. (J. Am. Chem. Soc. 103:3185-3191, 1981); automated synthesismethods; or the solid support method of U.S. Pat. No. 4,458,066,entitled “PROCESS FOR PREPARING POLYNUCLEOTIDES,” issued Jul. 3, 1984 toCaruthers et al., or other methods known to those skilled in the art.All of these references are incorporated by reference.

The term “primer” as used herein refers to a polynucleotide capable ofacting as a point of initiation of template-directed nucleic acidsynthesis when placed under conditions in which polynucleotide extensionis initiated (e.g., under conditions comprising the presence ofrequisite nucleoside triphosphates (as dictated by the template that iscopied) and a polymerase in an appropriate buffer and at a suitabletemperature or cycle(s) of temperatures (e.g., as in a polymerase chainreaction)). To further illustrate, primers can also be used in a varietyof other oligonuceotide-mediated synthesis processes, including asinitiators of de novo RNA synthesis and in vitro transcription-relatedprocesses (e.g., nucleic acid sequence-based amplification (NASBA),transcription mediated amplification (TMA), etc.). A primer is typicallya single-stranded oligonucleotide (e.g., oligodeoxyribonucleotide). Theappropriate length of a primer depends on the intended use of the primerbut typically ranges from 6 to 40 nucleotides, more typically from 15 to35 nucleotides. Short primer molecules generally require coolertemperatures to form sufficiently stable hybrid complexes with thetemplate. A primer need not reflect the exact sequence of the templatebut must be sufficiently complementary to hybridize with a template forprimer elongation to occur. In certain embodiments, the term “primerpair” means a set of primers including a 5′ sense primer (sometimescalled “forward”) that hybridizes with the complement of the 5′ end ofthe nucleic acid sequence to be amplified and a 3′ antisense primer(sometimes called “reverse”) that hybridizes with the 3′ end of thesequence to be amplified (e.g., if the target sequence is expressed asRNA or is an RNA). A primer can be labeled, if desired, by incorporatinga label detectable by spectroscopic, photochemical, biochemical,immunochemical, or chemical means. For example, useful labels include³²P, fluorescent dyes, electron-dense reagents, enzymes (as commonlyused in ELISA assays), biotin, or haptens and proteins for whichantisera or monoclonal antibodies are available.

The term “conventional” or “natural” when referring to nucleic acidbases, nucleoside triphosphates, or nucleotides refers to those whichoccur naturally in the polynucleotide being described (i.e., for DNAthese are dATP, dGTP, dCTP and dTTP). Additionally, dITP, and7-deaza-dGTP are frequently utilized in place of dGTP and 7-deaza-dATPcan be utilized in place of dATP in in vitro DNA synthesis reactions,such as sequencing. Collectively, these may be referred to as dNTPs.

The term “unconventional” or “modified” when referring to a nucleic acidbase, nucleoside, or nucleotide includes modification, derivations, oranalogues of conventional bases, nucleosides, or nucleotides thatnaturally occur in a particular polynucleotide. Certain unconventionalnucleotides are modified at the 2′ position of the ribose sugar incomparison to conventional dNTPs. Thus, although for RNA the naturallyoccurring nucleotides are ribonucleotides (i.e., ATP, GTP, CTP, UTP,collectively rNTPs), because these nucleotides have a hydroxyl group atthe 2′ position of the sugar, which, by comparison is absent in dNTPs,as used herein, ribonucleotides are unconventional nucleotides assubstrates for DNA polymerases. As used herein, unconventionalnucleotides include, but are not limited to, compounds used asterminators for nucleic acid sequencing. Exemplary terminator compoundsinclude but are not limited to those compounds that have a 2′,3′ dideoxystructure and are referred to as dideoxynucleoside triphosphates. Thedideoxynucleoside triphosphates ddATP, ddTTP, ddCTP and ddGTP arereferred to collectively as ddNTPs. Additional examples of terminatorcompounds include 2′-PO₄ analogs of ribonucleotides (see, e.g., U.S.Application Publication Nos. 2005/0037991 and 2005/0037398, which areboth incorporated by reference). Other unconventional nucleotidesinclude phosphorothioate dNTPs ([α-S]dNTPs), 5′-[α-borano]-dNTPs,[α]-methyl-phosphonate dNTPs, and ribonucleoside triphosphates (rNTPs).Unconventional bases may be labeled with radioactive isotopes such as³²P, ³³P, or ³⁵S; fluorescent labels; chemiluminescent labels;bioluminescent labels; hapten labels such as biotin; or enzyme labelssuch as streptavidin or avidin. Fluorescent labels may include dyes thatare negatively charged, such as dyes of the fluorescein family, or dyesthat are neutral in charge, such as dyes of the rhodamine family, ordyes that are positively charged, such as dyes of the cyanine family.Dyes of the fluorescein family include, e.g., FAM, HEX, TET, JOE, NANand ZOE. Dyes of the rhodamine family include Texas Red, ROX, R110, R6G,and TAMRA. Various dyes or nucleotides labeled with FAM, HEX, TET, JOE,NAN, ZOE, ROX, R110, R6G, Texas Red and TAMRA are marketed byPerkin-Elmer (Boston, Mass.), Applied Biosystems (Foster City, Calif.),or Invitrogen/Molecular Probes (Eugene, Oreg.). Dyes of the cyaninefamily include Cy2, Cy3, Cy5, and Cy7 and are marketed by GE HealthcareUK Limited (Amersham Place, Little Chalfont, Buckinghamshire, England).

As used herein, “percentage of sequence identity” is determined bycomparing two optimally aligned sequences over a comparison window,wherein the portion of the sequence in the comparison window cancomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same. Sequences are“substantially identical” to each other if they have a specifiedpercentage of nucleotides or amino acid residues that are the same(e.g., at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, or at least 95% identity over a specified region)), when comparedand aligned for maximum correspondence over a comparison window, ordesignated region as measured using one of the following sequencecomparison algorithms or by manual alignment and visual inspection.Sequences are “substantially identical” to each other if they are atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, or at least 55% identical. These definitionsalso refer to the complement of a test sequence. Optionally, theidentity exists over a region that is at least about 50 nucleotides inlength, or more typically over a region that is 100 to 500 or 1000 ormore nucleotides in length.

The terms “similarity” or “percent similarity,” in the context of two ormore polypeptide sequences, refer to two or more sequences orsubsequences that have a specified percentage of amino acid residuesthat are either the same or similar as defined by a conservative aminoacid substitutions (e.g., 60% similarity, optionally 65%, 70%, 75%, 80%,85%, 90%, or 95% similar over a specified region), when compared andaligned for maximum correspondence over a comparison window, ordesignated region as measured using one of the following sequencecomparison algorithms or by manual alignment and visual inspection.Sequences are “substantially similar” to each other if they are at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, or at least 55% similar to each other. Optionally,this similarly exists over a region that is at least about 50 aminoacids in length, or more typically over a region that is at least about100 to 500 or 1000 or more amino acids in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters are commonly used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities or similarities for the test sequencesrelative to the reference sequence, based on the program parameters.

A “comparison window,” as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well known in the art. Optimal alignment of sequencesfor comparison can be conducted, for example, by the local homologyalgorithm of Smith and Waterman (Adv. Appl. Math. 2:482, 1970), by thehomology alignment algorithm of Needleman and Wunsch (J. Mol. Biol.48:443, 1970), by the search for similarity method of Pearson and Lipman(Proc. Natl. Acad. Sci. USA 85:2444, 1988), by computerizedimplementations of these algorithms (e.g., GAP, BESTFIT, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by manual alignment andvisual inspection (see, e.g., Ausubel et al., Current Protocols inMolecular Biology (1995 supplement)).

Examples of an algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (Nuc. Acids Res.25:3389-402, 1977), and Altschul et al. (J. Mol. Biol. 215:403-10,1990), respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information (seethe internet at www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0) and N (penalty score for mismatchingresidues; always <0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, Proc.Natl. Acad. Sci. USA 90:5873-87, 1993). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, typically less thanabout 0.01, and more typically less than about 0.001.

The term “reverse transcription efficiency” refers to the fraction ofRNA molecules that are reverse transcribed as cDNA in a given reversetranscription reaction. In certain embodiments, the mutant DNApolymerases of the invention have improved reverse transcriptionefficiencies relative to unmodified forms of these DNA polymerases. Thatis, these mutant DNA polymerases reverse transcribe a higher fraction ofRNA templates than their unmodified forms under a particular set ofreaction conditions. Without being limited by theory, the ability of amutant DNA polymerase described herein to reverse transcribe a higherfraction of RNA templates can be due to an increased reversetranscription activity, for example, an increased nucleotideincorporation rate and/or increased processivity of the enzyme. Reversetranscription efficiency can be measured, for example, by measuring thecrossing point (Cp) of a PCR reaction using a RNA template, andcomparing the Cp value to a Cp value of a control reaction in which aDNA template of the same sequence (except U's are replaced with T's) isamplified, wherein the RNA and DNA amplifications use a common primerset and the same polymerase, e.g., as described in the examples. A testpolymerase has improved RT efficiency when the test polymerase has adecreased Cp value compared to a control polymerase when RNA is used asa template, but has a substantially unchanged Cp value relative to thecontrol polymerase when DNA is used as a template. In some embodiments apolymerase of the invention has an improved RT efficiency such that theCp is at least one, two, three, four, five, six, seven, eight, nine, tenor more units less than the corresponding control polymerase on the RNAtemplate. Improved RT efficiency of a test polymerase can be measured asdescribed in the Examples.

The term “mismatch tolerance” refers to the ability of a polymerase totolerate a mismatch-containing sequence when extending a nucleic acid(e.g., a primer or other oligonucleotide) in a template-dependent mannerby attaching (e.g., covalently) one or more nucleotides to the nucleicacid. The term “3′ mismatch tolerance” refers to the ability of apolymerase to tolerate a mismatch-containing (nearly complementary)sequence where the nucleic acid to be extended (e.g., a primer or otheroligonucleotide) has a mismatch with its template at the 3′ terminalnucleotide of the primer. Mismatches to the template may also be locatedat the 3′ penultimate nucleotide of the primer, or at another positionwithin the sequence of the primer.

The term “mismatch discrimination” refers to the ability of a polymeraseto distinguish a fully complementary sequence from a mismatch-containingsequence when extending a nucleic acid (e.g., a primer or otheroligonucleotide) in a template-dependent manner by attaching (e.g.,covalently) one or more nucleotides to the nucleic acid. The term“3′-mismatch discrimination” refers to the ability of a polymerase todistinguish a fully complementary sequence from a mismatch-containing(nearly complementary) sequence where the nucleic acid to be extended(e.g., a primer or other oligonucleotide) has a mismatch at the nucleicacid's 3′ terminus compared to the template to which the nucleic acidhybridizes. The term “mismatch” refers to the existence of one or morebase mispairings (or “noncomplementary base oppositions”) within astretch of otherwise complementary duplex-forming (or potentiallyduplex-forming) sequences.

The term “Cp value” or “crossing point” value refers to a value thatallows quantification of input target nucleic acids. The Cp value can bedetermined according to the second-derivative maximum method (VanLuu-The, et al., “Improved real-time RT-PCR method for high-throughputmeasurements using second derivative calculation and double correction,”BioTechniques, Vol. 38, No. 2, February 2005, pp. 287-293). In thesecond derivative method, a Cp corresponds to the first peak of a secondderivative curve. This peak corresponds to the beginning of a log-linearphase. The second derivative method calculates a second derivative valueof the real-time fluorescence intensity curve, and only one value isobtained. The original Cp method is based on a locally defined,differentiable approximation of the intensity values, e.g., by apolynomial function. Then the third derivative is computed. The Cp valueis the smallest root of the third derivative. The Cp can also bedetermined using the fit point method, in which the Cp is determined bythe intersection of a parallel to the threshold line in the log-linearregion (Van Luu-The, et al., BioTechniques, Vol. 38, No. 2, February2005, pp. 287-293). The Cp value provided by the LightCycler instrumentoffered by Roche by calculation according to the second-derivativemaximum method.

The term “PCR efficiency” refers to an indication of cycle to cycleamplification efficiency. PCR efficiency is calculated for eachcondition using the equation: % PCR efficiency=(10^((−slope))−1)×100,wherein the slope was calculated by linear regression with the log copynumber plotted on the y-axis and Cp plotted on the x-axis. PCRefficiency can be measured using a perfectly matched or mismatchedprimer template.

The term “nucleic acid extension rate” refers the rate at which abiocatalyst (e.g., an enzyme, such as a polymerase, ligase, or the like)extends a nucleic acid (e.g., a primer or other oligonucleotide) in atemplate-dependent or template-independent manner by attaching (e.g.,covalently) one or more nucleotides to the nucleic acid. To illustrate,certain mutant DNA polymerases described herein have improved nucleicacid extension rates relative to unmodified forms of these DNApolymerases, such that they can extend primers at higher rates thanthese unmodified forms under a given set of reaction conditions.

The term “tolerance of RT and polymerase inhibitors” refers to theability of a polymerase to maintain activity (polymerase or reversetranscription activity) in the presence of an amount of an inhibitorthat would inhibit the polymerase activity or reverse transcriptionactivity of a control polymerase. In some embodiments, the improvedpolymerase is capable of polymerase or reverse transcription activity inthe presence of an amount of the inhibitor that would essentiallyeliminate the control polymerase activity.

The term “5′-nuclease probe” refers to an oligonucleotide that comprisesat least one light emitting labeling moiety and that is used in a5′-nuclease reaction to effect target nucleic acid detection. In someembodiments, for example, a 5′-nuclease probe includes only a singlelight emitting moiety (e.g., a fluorescent dye, etc.). In certainembodiments, 5′-nuclease probes include regions of self-complementaritysuch that the probes are capable of forming hairpin structures underselected conditions. To further illustrate, in some embodiments a5′-nuclease probe comprises at least two labeling moieties and emitsradiation of increased intensity after one of the two labels is cleavedor otherwise separated from the oligonucleotide. In certain embodiments,a 5′-nuclease probe is labeled with two different fluorescent dyes,e.g., a 5′ terminus reporter dye and the 3′ terminus quencher dye ormoiety. In some embodiments, 5′-nuclease probes are labeled at one ormore positions other than, or in addition to, terminal positions. Whenthe probe is intact, energy transfer typically occurs between the twofluorophores such that fluorescent emission from the reporter dye isquenched at least in part. During an extension step of a polymerasechain reaction, for example, a 5′-nuclease probe bound to a templatenucleic acid is cleaved by the 5′ to 3′ nuclease activity of, e.g., aTaq polymerase or another polymerase having this activity such that thefluorescent emission of the reporter dye is no longer quenched.Exemplary 5′-nuclease probes are also described in, e.g., U.S. Pat. No.5,210,015, entitled “Homogeneous assay system using the nucleaseactivity of a nucleic acid polymerase,” issued May 11, 1993 to Gelfandet al., U.S. Pat. No. 5,994,056, entitled “Homogeneous methods fornucleic acid amplification and detection,” issued Nov. 30, 1999 toHiguchi, and U.S. Pat. No. 6,171,785, entitled “Methods and devices forhomogeneous nucleic acid amplification and detector,” issued Jan. 9,2001 to Higuchi, which are each incorporated by reference herein. Inother embodiments, a 5′ nuclease probe may be labeled with two or moredifferent reporter dyes and a 3′ terminus quencher dye or moiety.

The term “FRET” or “fluorescent resonance energy transfer” or “Foersterresonance energy transfer” refers to a transfer of energy between atleast two chromophores, a donor chromophore and an acceptor chromophore(referred to as a quencher). The donor typically transfers the energy tothe acceptor when the donor is excited by light radiation with asuitable wavelength. The acceptor typically re-emits the transferredenergy in the form of light radiation with a different wavelength. Whenthe acceptor is a “dark” quencher, it dissipates the transferred energyin a form other than light. Whether a particular fluorophore acts as adonor or an acceptor depends on the properties of the other member ofthe FRET pair. Commonly used donor-acceptor pairs include the FAM-TAMRApair. Commonly used quenchers are DABCYL and TAMRA. Commonly used darkquenchers include BlackHole Quenchers™ (BHQ), (Biosearch Technologies,Inc., Novato, Cal.), Iowa Black™ (Integrated DNA Tech., Inc.,Coralville, Iowa), and BlackBerry™ Quencher 650 (BBQ-650) (Berry &Assoc., Dexter, Mich.).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an amino acid sequence alignment of a region from thepolymerase domain of exemplary DNA polymerases from various species ofbacteria: Thermus species Z05 (Z05) (SEQ ID NO:12), Thermus aquaticus(Taq) (SEQ ID NO:13), Thermus filiformus (Tfi) (SEQ ID NO:14), Thermusflavus (Tfl) (SEQ ID NO:15), Thermus species sps17 (Sps17) (SEQ IDNO:16), Thermus thermophilus (Tth) (SEQ ID NO:17), Thermus caldophilus(Tca) (SEQ ID NO:18), Thermotoga maritima (Tma) (SEQ ID NO:19),Thermotoga neopolitana (Tne) (SEQ ID NO:20), Thermosipho africanus (Taf)(SEQ ID NO:21), Deinococcus radiodurans (Dra) (SEQ ID NO:23), Bacillusstearothermophilus (Bst) (SEQ ID NO:24), and Bacillus caldotenax (Bca)(SEQ ID NO:25). In addition, the polypeptide regions shown comprise theamino acid motif X₁-X₂-X₃-F-X₄-X₅-X₆-X₇-D-X₈-H-T-X₉-T-A-X₁₀-X₁₁ (SEQ IDNO:26), the variable positions of which are further defined herein. Thismotif is highlighted in bold type for each polymerase sequence. Aminoacid positions amenable to mutation in accordance with the presentinvention are indicated with an asterisk (*). Gaps in the alignments areindicated with a dot (.).

FIG. 2 provides sequence identities among the following DNA Polymerase Ienzymes: Thermus sp. Z05 DNA polymerase (Z05); Thermus aquaticus DNApolymerase (Taq); Thermus filiformis DNA polymerase (Tfi); Thermusflavus DNA polymerase (Tfl); Thermus sp. sps17 DNA polymerase (Sps17);Thermus thermophilus DNA polymerase (Tth); Thermus caldophilus DNApolymerase (Tca); Deinococcus radiodurans DNA polymerase (Dra);Thermotoga maritima DNA polymerase (Tma); Thermotoga neopolitana DNApolymerase (Tne); Thermosipho africanus DNA polymerase (Taf); Bacillusstearothermophilus DNA polymerase (Bst); and Bacillus caldotenax DNApolymerase (Bca). (A) sequence identities over the entire polymerase Ienzyme (corresponding to amino acids 1-834 of Z05); and (B) sequenceidentities over the polymerase sub domain corresponding to amino acids420-834 of Z05.

FIG. 3 provides sequence identities among various Thermus sp DNAPolymerase I enzymes: Thermus sp. Z05 DNA polymerase (Z05); Thermusaquaticus DNA polymerase (Taq); Thermus filiformis DNA polymerase (Tfi);Thermus flavus DNA polymerase (Tfl); Thermus sp. sps17 DNA polymerase(Sps17); Thermus thermophilus DNA polymerase (Tth); and Thermuscaldophilus DNA polymerase (Tca). (A) sequence identities over theentire polymerase I enzyme (corresponding to amino acids 1-834 of Z05);and (B) sequence identities over the polymerase sub domain correspondingto amino acids 420-834 of Z05.

DETAILED DESCRIPTION

The present invention provides improved DNA polymerases in which one ormore amino acids in the polymerase domain have been mutated relative toa functional DNA polymerase. The DNA polymerases of the invention areactive enzymes having increased reverse transcriptase efficiency (e.g.,in the presence of Mn²⁺ and Mg²⁺ divalent cations) relative to theunmodified form of the polymerase and/or increased mismatch tolerance,extension rate and tolerance of RT and polymerase inhibitors. In certainembodiments, the mutant DNA polymerases may be used at lowerconcentrations for superior or equivalent performance as the parentenzymes. In some embodiments, the mutant DNA polymerases have increasedreverse transcriptase efficiency while retaining substantially the sameDNA-dependent polymerase activity relative to an unmodified or controlpolymerase.

DNA polymerases that more efficiently perform reverse transcription arehelpful, for example, in a variety of applications involving assays thatemploy RT-PCR to detect and/or quantify RNA targets. The DNA polymerasesare therefore useful in a variety of applications involvingpolynucleotide extension as well as reverse transcription oramplification of polynucleotide templates, including, for example,applications in recombinant DNA studies and medical diagnosis ofdisease. The mutant DNA polymerases are also particularly useful,because of their tolerance for mis-matches, for detecting targets thatpossibly have variable sequences (e.g., viral targets, or cancer andother disease genetic markers).

In some embodiments, DNA polymerases of the invention can becharacterized by having the following motif:

-   -   X₁-X₂-X₃-Phe-X₄-X₅-X₆-X₇-Asp-X₈-His-Thr-X₉-Thr-Ala-X₁₀-X₁₁ (also        referred to herein in the one-letter code as        X₁-X₂-X₃-F-X₄-X₅-X₆-X₇-D-X₈-H-T-X₉-T-A-X₁₀-X₁₁ (SEQ ID NO:8);    -   wherein:    -   X₁ is Ile (I), Leu (L), Val (V), Gln (O) or Met (M);    -   X₂ is Arg (R), Lys (K), Gln (O), or Glu (E);    -   X₃ is Val (V) or Ala (A);    -   X₄ is Gln (O), Arg (R), Glu (E), Lys (K) or Val (V);    -   X₅ is Glu (E) or Arg (R);    -   X₆ is Gly (G) or Asp (D);    -   X₇ is Lys (K), Arg (R), Ile (I), Leu (L), Ala (A);    -   X₈ is any amino acid other than Ile (I) or Val (V);    -   X₉ is Gln (O), Glu (E), Leu (L), Ile (I), Arg (R) or Lys (K);    -   X₁₀ is Ser (S), Ala (A) or Met (M);    -   X₁₁ is Trp (W), Arg (R), Lys (K), Gln (O) or Asp (D).

In some embodiments, X₈ is selected from G, A, W, P, S, T, F, Y, C, N,Q, D, E, K, R, L, M, or H.

In some embodiments, DNA polymerases of the invention can becharacterized by having the following motif:

-   -   Ile-Arg-Val-Phe-X₄-Glu-Gly-X₇-Asp-X₈-His-Thr-X₉-Thr-Ala-X₁₀-Trp        (also referred to herein in the one-letter code as        I-R-V-F-X₄-E-G-X₇-D-X₈-H-T-X₉-T-A-X₁₀-W (SEQ ID NO:9);    -   wherein:    -   X₄ is Gln (O) or Arg (R);    -   X₇ is Lys (K) or Arg (R);    -   X₈ is any amino acid other than Ile (I);    -   X₉ is Gln (O) or Glu (E);    -   X₁₀ is Ser (S) or Ala (A).

In some embodiments, DNA polymerases of the invention can becharacterized by having the following motif:

-   -   Ile-Arg-Val-Phe-Gln-Glu-Gly-Lys-Asp-X₈-His-Thr-Gln-Thr-Ala-Ser-Trp        (also referred to herein in the one-letter code as        I—R-V-F-Q-E-G-K-D-X₈-H-T-Q-T-A-S-W (SEQ ID NO:10);    -   wherein:    -   X₈ is any amino acid other than Ile (I).

In some embodiments, DNA polymerases of the invention can becharacterized by having the following motif:

-   -   Ile-Arg-Val-Phe-Gln-Glu-Gly-Lys-Asp-X₈-His-Thr-Gln-Thr-Ala-Ser-Trp        (also referred to herein in the one-letter code as        I—R-V-F-Q-E-G-K-D-X₈-H-T-Q-T-A-S-W (SEQ ID NO:11);    -   wherein:    -   X₈ is Phe (F).

In some embodiments, DNA polymerases of the invention can becharacterized by having the above motifs (e.g., SEQ ID NOs:8, 9, 10, and11), optionally in combination with additional motifs described below.For example, in some embodiments, the DNA polymerase further comprisesthe motif of SEQ ID NO:29 and/or SEQ ID NO:38.

This motif is present within the “fingers” domain (N alpha helix) ofmany Family A type DNA-dependent DNA polymerases, particularlythermostable DNA polymerases from thermophilic bacteria (Li et al., EMBOJ. 17:7514-7525, 1998). For example, FIG. 1 shows an amino acid sequencealignment of a region from the “fingers” domain of DNA polymerases fromseveral species of bacteria: Bacillus caldotenax, Bacillusstearothermophilus, Deinococcus radiodurans, Thermosipho africanus,Thermotoga maritima, Thermotoga neopolitana, Thermus aquaticus, Thermuscaldophilus, Thermus filiformus, Thermus flavus, Thermus sp. sps17,Thermus sp. Z05, and Thermus thermophilus. As shown, the native sequencecorresponding to the motif above is present in each of thesepolymerases, indicating a conserved function for this region of thepolymerase. FIG. 2 provides sequence identities among these DNApolymerases.

Accordingly, in some embodiments, the invention provides for apolymerase comprising SEQ ID NO:8, 9, 10, or 11, having the improvedactivity and/or characteristics described herein, and wherein the DNApolymerase is otherwise a wild-type or a naturally occurring DNApolymerase, such as, for example, a polymerase from any of the speciesof thermophilic bacteria listed above, or is substantially identical tosuch a wild-type or a naturally occurring DNA polymerase. For example,in some embodiments, the polymerase of the invention comprises SEQ IDNO:8, 9, 10, or 11 and is at least 80%, 85%, 90%, or 95% identical toSEQ ID NO:1, 2, 3, 4, 5, 6, 7, 32, 33, 34, 35, 36, 37, or 39. In onevariation, the unmodified form of the polymerase is from a species ofthe genus Thermus. In other embodiments of the invention, the unmodifiedpolymerase is from a thermophilic species other than Thermus, e.g.,Thermotoga. The full nucleic acid and amino acid sequence for numerousthermostable DNA polymerases are available. The sequences each ofThermus aquaticus (Taq) (SEQ ID NO:2), Thermus thermophilus (Tth) (SEQID NO:6), Thermus species Z05 (SEQ ID NO:1), Thermus species sps17 (SEQID NO:5), Thermotoga maritima (Tma) (SEQ ID NO:34), and Thermosiphoafricanus (Taf) (SEQ ID NO:33) polymerase have been published in PCTInternational Patent Publication No. WO 92/06200, which is incorporatedherein by reference. The sequence for the DNA polymerase from Thermusflavus (SEQ ID NO:4) has been published in Akhmetzjanov and Vakhitov(Nucleic Acids Research 20:5839, 1992), which is incorporated herein byreference. The sequence of the thermostable DNA polymerase from Thermuscaldophilus (SEQ ID NO:7) is found in EMBL/GenBank Accession No. U62584.The sequence of the thermostable DNA polymerase from Thermus filiformiscan be recovered from ATCC Deposit No. 42380 using, e.g., the methodsprovided in U.S. Pat. No. 4,889,818, as well as the sequence informationprovided in Table 1. The sequence of the Thermotoga neapolitana DNApolymerase (SEQ ID NO:35) is from GeneSeq Patent Data Base Accession No.R98144 and PCT WO 97/09451, each incorporated herein by reference. Thesequence of the thermostable DNA polymerase from Bacillus caldotenax(SEQ ID NO:37 is described in, e.g., Uemori et al. (J Biochem (Tokyo)113(3):401-410, 1993; see also, Swiss-Prot database Accession No. Q04957and GenBank Accession Nos. D12982 and BAA02361), which are eachincorporated by reference. Examples of unmodified forms of DNApolymerases that can be modified as described herein are also describedin, e.g., U.S. Pat. No. 6,228,628, entitled “Mutant chimeric DNApolymerase” issued May 8, 2001 to Gelfand et al.; U.S. Pat. No.6,346,379, entitled “Thermostable DNA polymerases incorporatingnucleoside triphosphates labeled with fluorescein family dyes” issuedFeb. 12, 2002 to Gelfand et al.; U.S. Pat. No. 7,030,220, entitled“Thermostable enzyme promoting the fidelity of thermostable DNApolymerases-for improvement of nucleic acid synthesis and amplificationin vitro” issued Apr. 18, 2006 to Ankenbauer et al.; U.S. Pat. No.6,881,559 entitled “Mutant B-type DNA polymerases exhibiting improvedperformance in PCR” issued Apr. 19, 2005 to Sobek et al.; U.S. Pat. No.6,794,177 entitled “Modified DNA-polymerase from carboxydothermushydrogenoformans and its use for coupled reverse transcription andpolymerase chain reaction” issued Sep. 21, 2004 to Markau et al.; U.S.Pat. No. 6,468,775, entitled “Thermostable DNA polymerase fromcarboxydothermus hydrogenoformans” issued Oct. 22, 2002 to Ankenbauer etal.; and U.S. Pat. Nos. 7,148,049 entitled “Thermostable or thermoactiveDNA polymerase molecules with attenuated 3′-5′ exonuclease activity”issued Dec. 12, 2006 to Schoenbrunner et al.; U.S. Pat. No. 7,179,590entitled “High temperature reverse transcription using mutant DNApolymerases” issued Feb. 20, 2007 to Smith et al.; U.S. Pat. No.7,410,782 entitled “Thermostable enzyme promoting the fidelity ofthermostable DNA polymerases-for improvement of nucleic acid synthesisand amplification in vitro” issued Aug. 12, 2008 to Ankenbauer et al.;U.S. Pat. No. 7,378,262 entitled “Reversibly modified thermostableenzymes for DNA synthesis and amplification in vitro” issued May 27,2008 to Sobek et al., which are each incorporated by reference.Representative full length polymerase sequences are also provided in thesequence listing.

Also amenable to the mutations described herein are functional DNApolymerases that have been previously modified (e.g., by amino acidsubstitution, addition, or deletion). In some embodiments, suchfunctional modified polymerases retain the amino acid motif of SEQ IDNO:8 (or a motif of SEQ ID NO:9, 10 or 11), and optionally the aminoacid motif of SEQ ID NO:38. Thus, suitable unmodified DNA polymerasesalso include functional variants of wild-type or naturally occurringpolymerases. Such variants typically will have substantial sequenceidentity or similarity to the wild-type or naturally occurringpolymerase, typically at least 80% sequence identity and more typicallyat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity.

In some embodiments, the polymerase of the invention, as well as havinga polymerase domain comprising SEQ ID NOS:8, 9, 10, or 11 also comprisesa nuclease domain (e.g., corresponding to positions 1 to 291 of Z05)

In some embodiments, a polymerase of the invention is a chimericpolymerase, i.e., comprising polypeptide regions from two or moreenzymes. Examples of such chimeric DNA polymerases are described in,e.g., U.S. Pat. No. 6,228,628, which is incorporated by reference hereinin its entirety. Particularly suitable are chimeric CS-family DNApolymerases, which include the CS5 (SEQ ID NO:27) and CS6 (SEQ ID NO:28)polymerases and variants thereof having substantial amino acid sequenceidentity or similarity to SEQ ID NO:27 or SEQ ID NO:28 (typically atleast 80% amino acid sequence identity and more typically at least 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequenceidentity) and can thus be modified to contain SEQ ID NO:8. The CS5 andCS6 DNA polymerases are chimeric enzymes derived from Thermus sp. Z05and Thermotoga maritima (Tma) DNA polymerases. They comprise theN-terminal 5′-nuclease domain of the Thermus enzyme and the C-terminal3′-5′ exonuclease and the polymerase domains of the Tma enzyme. Theseenzymes have efficient reverse transcriptase activity, can extendnucleotide analog-containing primers, and can incorporatealpha-phosphorothioate dNTPs, dUTP, dITP, and also fluorescein- andcyanine-dye family labeled dNTPs. The CS5 and CS6 polymerases are alsoefficient Mg²⁺-activated PCR enzymes. The CS5 and CS6 chimericpolymerases are further described in, e.g., U.S. Pat. No. 7,148,049,which is incorporated by reference herein in its entirety.

In some embodiments, the amino acid substitutions are single amino acidsubstitutions. The DNA polymerases provided herein can comprise one ormore amino acid substitutions in the active site relative to theunmodified polymerase. In some embodiments, the amino acidsubstitution(s) comprise at least position X₈ of the motif set forth inSEQ ID NO:8 (or a motif of SEQ ID NO:9, 10 or 11). Amino acidsubstitution at this position confers increased reverse transcriptaseefficiency, mismatch tolerance, extension rate and/or tolerance of RTand polymerase inhibitors, yielding a mutant DNA polymerase with anincreased reverse transcriptase efficiency, mismatch tolerance,extension rate and/or tolerance of RT and polymerase inhibitors relativeto the unmodified polymerase. Typically, the amino acid at position X₈is substituted with an amino acid that does not correspond to the nativesequence within the motif set forth in SEQ ID NO:8 (or a motif of SEQ IDNO:9, 10 or 11). Thus, typically, the amino acid at position X₈, ifsubstituted, is not Ile (I) or Val (V), as I or V occurs at thisposition in naturally-occurring polymerases. See, e.g., FIG. 1. Incertain embodiments, amino acid substitutions include G, A, W, P, S, T,F, Y, C, N, Q, D, E, K, R, L, M, or H at position X₈. In certainembodiments, amino acid substitutions include Phenylalanine (F) atposition X₈. Other suitable amino acid substitution(s) at one or more ofthe identified sites can be determined using, e.g., known methods ofsite-directed mutagenesis and determination of polynucleotide extensionperformance in assays described further herein or otherwise known topersons of skill in the art.

In some embodiments, the polymerase of the invention comprises SEQ IDNO:8, 9, 10, or 11 and further comprises one or more additional aminoacid changes (e.g., by amino acid substitution, addition, or deletion)compared to a native polymerase. In some embodiments, such polymerasesretain the amino acid motif of SEQ ID NO:8 (or a motif of SEQ ID NO:9,10 or 11), and further comprise the amino acid motif of SEQ ID NO:38(corresponding to the D580X mutation of Z05 (SEQ ID NO:1)) as follows:

-   -   Thr-Gly-Arg-Leu-Ser-Ser-X_(b7)-X_(b8)-Pro-Asn-Leu-Gln-Asn (also        referred to herein in the one-letter code as        T-G-R-L-S-S-X_(b7)-X_(b8)-P-N-L-Q-N) (SEQ ID NO:38); wherein    -   X_(b7) is Ser (S) or Thr (T); and    -   X_(b8) is any amino acid other than Asp (D) or Glu (E)        The mutation characterized by SEQ ID NO:38 is discussed in more        detail in, e.g., US Patent Publication No. 2009/0148891. Such        functional variant polymerases typically will have substantial        sequence identity or similarity to the wild-type or naturally        occurring polymerase (e.g., SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 32,        33, 34, 35, 36, 37, or 39), typically at least 80% amino acid        sequence identity and more typically at least 90%, 91%, 92%,        93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence        identity.

In some embodiments, the polymerase of the invention comprises SEQ IDNO:8, 9, 10, or 11 and further comprises the amino acid motif of SEQ IDNO:29 (corresponding to the I709X mutation of Z05 (SEQ ID NO:1) asfollows:

-   -   X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-Gly-Tyr-Val-X₁₄-Thr-Leu        (also referred to herein in the one-letter code as        X₁-X₂-X₃-X_(a4)-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-G-Y-V-X₁₄-T-L)        (SEQ ID NO:29); wherein        -   X₁ is Ala (A), Asp (D), Ser (S), Glu (E), Arg (R) or Gln            (O);        -   X₂ is Trp (W) or Tyr (Y);        -   X₃ is any amino acid other than Ile (I), Leu (L) or Met (M);        -   X₄ is Glu (E), Ala (A), Gln (O), Lys (K), Asn (N) or Asp            (D);        -   X₅ is Lys (K), Gly (G), Arg (R), Gln (O), H is (H) or Asn            (N);        -   X₆ is Thr (T), Val (V), Met (M) or Ile (I);        -   X₇ is Leu (L), Val (V) or Lys (K);        -   X₈ is Glu (E), Ser (S), Ala (A), Asp (D) or Gln (O);        -   X₉ is Glu (E) or Phe (F);        -   X₁₀ is Gly (G) or Ala (A);        -   X₁₁ is Arg (R) or Lys (K);        -   X₁₂ is Lys (K), Arg (R), Glu (E), Thr (T) or Gln (O);        -   X₁₃ is Arg (R), Lys (K) or H is (H); and        -   X₁₄ is Glu (E), Arg (R) or Thr (T).

In some embodiments, such functional variant polymerases typically willhave substantial sequence identity or similarity to the wild-type ornaturally occurring polymerase (e.g., SEQ ID NO: 1, 2, 3, 4, 5, 6, 7,32, 33, 34, 35, 36, 37, or 39), typically at least 80% amino acidsequence identity and more typically at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% amino acid sequence identity.

In some embodiments, the DNA polymerase of the invention comprises anamino acid substitution at position X₈ (e.g., as in a motif selectedfrom SEQ ID NO:8, 9, 10 or 11) and comprises an amino acid substitutioncorresponding to SEQ ID NO:38 and SEQ ID NO:29.

In some embodiments, the amino acid at position X₈ is substituted withan amino acid as set forth in SEQ ID NO:8, 9, 10 or 11, and the aminoacid at position X_(b8) is substituted with an amino acid as set forthin SEQ ID NO:38. Thus, in some embodiments, the amino acid at positionX₈ is any amino acid other than Ile (I) and the amino acid at positionX_(b8) is any amino acid other than Asp (D) or Glu (E). In someembodiments, amino acid substitutions include Leucine (L), Glycine (G),Threonine (T), Glutamine (Q), Alanine (A), Serine (S), Asparagine (N),Arginine (R), and Lysine (K) at position X_(b8) of SEQ ID NO:38. Incertain embodiments, amino acid substitutions independently includeMethionine (M) at position X₈ of SEQ ID NO:8, 9, 10 or 11, and Glycine(G) at position X_(b8) of SEQ ID NO:38.

In some embodiments, the amino acid at position X₈ is substituted withan amino acid as set forth in SEQ ID NO:8, 9, 10 or 11, and the aminoacid at position X₃ (of SEQ ID NO:29) is substituted with an amino acidas set forth in SEQ ID NO:29. Thus, in some embodiments, the amino acidat position X₈ is any amino acid other than Ile (I) and the amino acidat position X₃ is any amino acid other than Ile (I), Leu (L) or Met (M).In some embodiments, amino acid substitutions include Lysine (K),Arginine (R), Serine (S), Glycine (G) or Alanine (A) at position X₃ ofSEQ ID NO:29. In certain embodiments, amino acid substitutionsindependently include Methionine (M) at position X₈ of SEQ ID NO:8, 9,10 or 11, and Lysine (K) at position X₃ of SEQ ID NO:29.

Other suitable amino acid substitution(s) at one or more of theidentified sites can be determined using, e.g., known methods ofsite-directed mutagenesis and determination of polynucleotide extensionperformance in assays described further herein or otherwise known topersons of skill in the art, e.g., amino acid substitutions described inU.S. Pat. Application Publication Nos. 2009/0148891 and 2009/0280539,which are incorporated by reference herein in its entirety.

Because the precise length of DNA polymerases vary, the precise aminoacid positions corresponding to each of X₈ (SEQ ID NO:8), X_(b8) (SEQ IDNO:38) and X₃ (SEQ ID NO:29) can vary depending on the particular mutantpolymerase used. Amino acid and nucleic acid sequence alignment programsare readily available (see, e.g., those referred to supra) and, giventhe particular motifs identified herein, serve to assist in theidentification of the exact amino acids (and corresponding codons) formodification in accordance with the present invention. The positionscorresponding to each of X₈, X_(b8) and X₃ are shown in Table 1 forrepresentative chimeric thermostable DNA polymerases and thermostableDNA polymerases from exemplary thermophilic species.

TABLE 1 Amino Acid Positions Corresponding to Motif Positions X₈ (e.g.,of SEQ ID NOs: 8, 9, 10, and 11), X_(b8) (of SEQ ID NO: 38) and X₃ (ofSEQ ID NO: 29) in Exemplary Polymerases. Amino Acid Position Organism orChimeric Sequence X_(b8) (of SEQ ID X₃ (of SEQ ID Consensus (SEQ ID NO:)X₈ NO: 38) NO: 29) T. thermophilus (6) 640 580 709 T. caldophilus (7)640 580 709 T. sp. Z05 (1) 640 580 709 T. aquaticus (2) 638 578 707 T.flavus (4) 637 577 706 T. filiformis (3) 636 576 705 T. sp. sps17 (5)636 576 705 T. maritima (34) 701 640 770 T. neapolitana (35) 701 640 770T. africanus (33) 700 639 769 B. caldotenax (37) 682 621 751 B.stearothermophilus (36) 681 620 750 CS5 (27) 701 640 770 CS6 (28) 701640 770

In some embodiments, the DNA polymerase of the present invention isderived from Thermus sp. Z05 DNA polymerase (SEQ ID NO:1) or a variantthereof (e.g., carrying the D580G mutation or the like). As referred toabove, in Thermus sp. Z05 DNA polymerase, position X₈ corresponds toIsoleucine (I) at position 640; position X_(b8) corresponds to Aspartate(D) at position 580, and position X₃ corresponds to Isoleucine (I) atposition 709. Thus, in certain variations of the invention, the mutantpolymerase comprises at least one amino acid substitution, relative to aThermus sp. Z05 DNA polymerase (or a DNA polymerase that issubstantially identical (e.g., at least about 60%, 65%, 70%, 75%, 80%,85%, 90%, or 95% identical) to SEQ ID NO:1), at I640, D580 and/or I709.Thus, typically, the amino acid at position 640 of SEQ ID NO:1 is not I.In some embodiments, the amino acid at position 640 of SEQ ID NO:1 isselected from G, A, V, R, F, W, P, S, T, C, Y, N, Q, D, E, K, L, M, orH. In certain embodiments, the amino acid residue at position 640 of SEQID NO:1 is F. In certain embodiments, amino acid residues at positionD580 of SEQ ID NO:1 can be selected from Leucine (L), Glycine (G),Threonine (T), Glutamine (Q), Alanine (A), Serine (S), Asparagine (N),Arginine (R), and Lysine (K). Thus, in some embodiments, the amino acidresidue at position 580 of SEQ ID NO:1 is Glycine (G). Further, incertain embodiments, the amino acid at position 709 of SEQ ID NO:1 isnot I. In some embodiments, the amino acid at position 709 of SEQ IDNO:1 is selected from G, A, V, R, F, W, P, S, T, C, Y, N, Q, D, E, K, L,M, or H. In some embodiments, the amino acid at position 709 of SEQ IDNO: 1 is K, R, S, G or A. In some embodiments, the amino acid atposition 709 of SEQ ID NO: 1 is K.

Exemplary Thermus sp. Z05 DNA polymerase mutants include thosecomprising the amino acid substitution(s) I640F, and/or I709K (or I709R,I709S, I709G, I709A), and/or D580G. In some embodiments, the mutantThermus sp. Z05 DNA polymerase comprises, e.g., amino acid residuesubstitutions I640F and D580G. In some embodiments, the mutant Thermussp. Z05 DNA polymerase comprises, e.g., amino acid residue substitutionsI640F and I709K. In some embodiments, the mutant Thermus sp. Z05 DNApolymerase comprises, e.g., amino acid residue substitutions I640F,I709K, and D580G. In certain embodiments, the mutant Thermus sp. Z05 DNApolymerase comprises, e.g., amino acid residue substitutionsindependently selected from I640F, I709K, and/or D580G.

In some embodiments, the amino acid corresponding to position 324 of SEQID NO:1 is Lys (K). In some embodiments, the amino acid corresponding toposition 324 of SEQ ID NO:1 is not Met (M). In some embodiments, theamino acid corresponding to position 461 of SEQ ID NO:1 is Leu (L). Insome embodiments, the amino acid corresponding to position 461 of SEQ IDNO:1 is not Met (M). In some embodiments, the amino acid correspondingto position 517 of SEQ ID NO:1 is Ser (S). In some embodiments, theamino acid corresponding to position 517 of SEQ ID NO:1 is not Arg (R).In some embodiments, the amino acid corresponding to position 741 of SEQID NO:1 is Ser (S). In some embodiments, the amino acid corresponding toposition 741 of SEQ ID NO:1 is not Gly (G). In some embodiments, theamino acid corresponding to position 775 of SEQ ID NO:1 is Arg (R). Insome embodiments, the amino acid corresponding to position 775 of SEQ IDNO:1 is not Gly (G). In some embodiments, the amino acid correspondingto position 791 of SEQ ID NO:1 is Leu (L). In some embodiments, theamino acid corresponding to position 789 of SEQ ID NO:1 is not Phe (F).

The inventors have shown that substitutions at the amino acidcorresponding to position 709 of SEQ ID NO:1 described above can resultin DNA polymerases having improved (i.e., increased) reversetranscription efficiency, increased RT-PCR activity (e.g., moreefficient amplification of an RNA template without compromising PCRefficiency on a DNA template), increased RT-PCR efficiency in thepresence of Mg²⁺, increased reverse transcriptase activity in thepresence of inhibitors (e.g., breakdown products of hemoglobin such ashemin, and/or heparin), increased extension rate and improved3′-mismatch tolerance compared to a control polymerase. See U.S. PatentApplication No. 61/474,160, filed Apr. 11, 2011, the contents of whichare incorporated by reference herein in its entirety. Thus, it isexpected that the improved polymerases that comprise substitutions atthe amino acid corresponding to position 709 of SEQ ID NO:1 describedherein will also have the improved properties described above.

In addition to the mutations and substitutions described herein, the DNApolymerases of the present invention can also include other,non-substitutional modification(s). Such modifications can include, forexample, covalent modifications known in the art to confer an additionaladvantage in applications comprising polynucleotide extension. Forexample, one such modification is a thermally reversible covalentmodification that inactivates the enzyme, but which is reversed toactivate the enzyme upon incubation at an elevated temperature, such asa temperature typically used for polynucleotide extension. Exemplaryreagents for such thermally reversible modifications are described inU.S. Pat. Nos. 5,773,258 and 5,677,152 to Birch et al., which areexpressly incorporated by reference herein in their entirety.

The DNA polymerases of the present invention can be constructed bymutating the DNA sequences that encode the corresponding unmodifiedpolymerase (e.g., a wild-type polymerase or a corresponding variant fromwhich the polymerase of the invention is derived), such as by usingtechniques commonly referred to as site-directed mutagenesis. Nucleicacid molecules encoding the unmodified form of the polymerase can bemutated by a variety of polymerase chain reaction (PCR) techniqueswell-known to one of ordinary skill in the art. (See, e.g., PCRStrategies (M. A. Innis, D. H. Gelfand, and J. J. Sninsky eds., 1995,Academic Press, San Diego, Calif.) at Chapter 14; PCR Protocols: A Guideto Methods and Applications (M. A. Innis, D. H. Gelfand, J. J. Sninsky,and T. J. White eds., Academic Press, NY, 1990).

By way of non-limiting example, the two primer system, utilized in theTransformer Site-Directed Mutagenesis kit from Clontech, may be employedfor introducing site-directed mutants into a polynucleotide encoding anunmodified form of the polymerase. Following denaturation of the targetplasmid in this system, two primers are simultaneously annealed to theplasmid; one of these primers contains the desired site-directedmutation, the other contains a mutation at another point in the plasmidresulting in elimination of a restriction site. Second strand synthesisis then carried out, tightly linking these two mutations, and theresulting plasmids are transformed into a mutS strain of E. coli.Plasmid DNA is isolated from the transformed bacteria, restricted withthe relevant restriction enzyme (thereby linearizing the unmutatedplasmids), and then retransformed into E. coli. This system allows forgeneration of mutations directly in an expression plasmid, without thenecessity of subcloning or generation of single-stranded phagemids. Thetight linkage of the two mutations and the subsequent linearization ofunmutated plasmids result in high mutation efficiency and allow minimalscreening. Following synthesis of the initial restriction site primer,this method requires the use of only one new primer type per mutationsite. Rather than prepare each positional mutant separately, a set of“designed degenerate” oligonucleotide primers can be synthesized inorder to introduce all of the desired mutations at a given sitesimultaneously. Transformants can be screened by sequencing the plasmidDNA through the mutagenized region to identify and sort mutant clones.Each mutant DNA can then be restricted and analyzed by electrophoresis,such as for example, on a Mutation Detection Enhancement gel(Mallinckrodt Baker, Inc., Phillipsburg, N.J.) to confirm that no otheralterations in the sequence have occurred (by band shift comparison tothe unmutagenized control). Alternatively, the entire DNA region can besequenced to confirm that no additional mutational events have occurredoutside of the targeted region.

DNA polymerases with more than one amino acid substituted can begenerated in various ways. In the case of amino acids located closetogether in the polypeptide chain, they may be mutated simultaneouslyusing one oligonucleotide that codes for all of the desired amino acidsubstitutions. If however, the amino acids are located some distancefrom each other (separated by more than ten amino acids, for example) itis more difficult to generate a single oligonucleotide that encodes allof the desired changes. Instead, one of two alternative methods may beemployed. In the first method, a separate oligonucleotide is generatedfor each amino acid to be substituted. The oligonucleotides are thenannealed to the single-stranded template DNA simultaneously, and thesecond strand of DNA that is synthesized from the template will encodeall of the desired amino acid substitutions. An alternative methodinvolves two or more rounds of mutagenesis to produce the desiredmutant. The first round is as described for the single mutants: DNAencoding the unmodified polymerase is used for the template, anoligonucleotide encoding the first desired amino acid substitution(s) isannealed to this template, and the heteroduplex DNA molecule is thengenerated. The second round of mutagenesis utilizes the mutated DNAproduced in the first round of mutagenesis as the template. Thus, thistemplate already contains one or more mutations. The oligonucleotideencoding the additional desired amino acid substitution(s) is thenannealed to this template, and the resulting strand of DNA now encodesmutations from both the first and second rounds of mutagenesis. Thisresultant DNA can be used as a template in a third round of mutagenesis,and so on. Alternatively, the multi-site mutagenesis method of Seyfang &Jin (Anal. Biochem. 324:285-291. 2004) may be utilized.

Accordingly, also provided are recombinant nucleic acids encoding any ofthe DNA polymerases of the present invention. Using a nucleic acid ofthe present invention, encoding a DNA polymerase, a variety of vectorscan be made. Any vector containing replicon and control sequences thatare derived from a species compatible with the host cell can be used inthe practice of the invention. Generally, expression vectors includetranscriptional and translational regulatory nucleic acid regionsoperably linked to the nucleic acid encoding the DNA polymerase. Theterm “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. In addition, the vector may contain a PositiveRetroregulatory Element (PRE) to enhance the half-life of thetranscribed mRNA (see Gelfand et al. U.S. Pat. No. 4,666,848). Thetranscriptional and translational regulatory nucleic acid regions willgenerally be appropriate to the host cell used to express thepolymerase. Numerous types of appropriate expression vectors, andsuitable regulatory sequences are known in the art for a variety of hostcells. In general, the transcriptional and translational regulatorysequences may include, e.g., promoter sequences, ribosomal bindingsites, transcriptional start and stop sequences, translational start andstop sequences, and enhancer or activator sequences. In typicalembodiments, the regulatory sequences include a promoter andtranscriptional start and stop sequences. Vectors also typically includea polylinker region containing several restriction sites for insertionof foreign DNA. In certain embodiments, “fusion flags” are used tofacilitate purification and, if desired, subsequent removal of tag/flagsequence, e.g., “His-Tag”. However, these are generally unnecessary whenpurifying a thermoactive and/or thermostable protein from a mesophilichost (e.g., E. coli) where a “heat-step” may be employed. Theconstruction of suitable vectors containing DNA encoding replicationsequences, regulatory sequences, phenotypic selection genes, and thepolymerase of interest are prepared using standard recombinant DNAprocedures. Isolated plasmids, viral vectors, and DNA fragments arecleaved, tailored, and ligated together in a specific order to generatethe desired vectors, as is well-known in the art (see, e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual (Cold Spring HarborLaboratory Press, New York, N.Y., 2nd ed. 1989)).

In certain embodiments, the expression vector contains a selectablemarker gene to allow the selection of transformed host cells. Selectiongenes are well known in the art and will vary with the host cell used.Suitable selection genes can include, for example, genes coding forampicillin and/or tetracycline resistance, which enables cellstransformed with these vectors to grow in the presence of theseantibiotics.

In one aspect of the present invention, a nucleic acid encoding a DNApolymerase is introduced into a cell, either alone or in combinationwith a vector. By “introduced into” or grammatical equivalents herein ismeant that the nucleic acids enter the cells in a manner suitable forsubsequent integration, amplification, and/or expression of the nucleicacid. The method of introduction is largely dictated by the targetedcell type. Exemplary methods include CaPO₄ precipitation, liposomefusion, LIPOFECTIN®, electroporation, viral infection, and the like.

In some embodiments, prokaryotes are typically used as host cells forthe initial cloning steps of the present invention. They areparticularly useful for rapid production of large amounts of DNA, forproduction of single-stranded DNA templates used for site-directedmutagenesis, for screening many mutants simultaneously, and for DNAsequencing of the mutants generated. Suitable prokaryotic host cellsinclude E. coli K12 strain 94 (ATCC No. 31,446), E. coli strain W3110(ATCC No. 27,325), E. coli K12 strain DG116 (ATCC No. 53,606), E. coliX1776 (ATCC No. 31,537), and E. coli B; however many other strains of E.coli, such as HB101, JM101, NM522, NM538, NM539, and many other speciesand genera of prokaryotes including bacilli such as Bacillus subtilis,other enterobacteriaceae such as Salmonella typhimurium or Serratiamarcesans, and various Pseudomonas species can all be used as hosts.Prokaryotic host cells or other host cells with rigid cell walls aretypically transformed using the calcium chloride method as described insection 1.82 of Sambrook et al., supra. Alternatively, electroporationcan be used for transformation of these cells. Prokaryote transformationtechniques are set forth in, for example Dower, in Genetic Engineering,Principles and Methods 12:275-296 (Plenum Publishing Corp., 1990);Hanahan et al., Meth. Enzymol., 204:63, 1991. Plasmids typically usedfor transformation of E. coli include pBR322, pUCI8, pUCI9, pUCI18,pUCI19, and Bluescript M13, all of which are described in sections1.12-1.20 of Sambrook et al., supra. However, many other suitablevectors are available as well.

The DNA polymerases of the present invention are typically produced byculturing a host cell transformed with an expression vector containing anucleic acid encoding the DNA polymerase, under the appropriateconditions to induce or cause expression of the DNA polymerase. Methodsof culturing transformed host cells under conditions suitable forprotein expression are well-known in the art (see, e.g., Sambrook etal., supra). Suitable host cells for production of the polymerases fromlambda pL promotor-containing plasmid vectors include E. coli strainDG116 (ATCC No. 53606) (see U.S. Pat. No. 5,079,352 and Lawyer, F. C. etal., PCR Methods and Applications 2:275-87, 1993, which are bothincorporated herein by reference). Following expression, the polymerasecan be harvested and isolated. Methods for purifying the thermostableDNA polymerase are described in, for example, Lawyer et al., supra. Oncepurified, the ability of the DNA polymerases to have improved RTefficiency, increased mis-match tolerance, extension rate and/ortolerance of RT and polymerase inhibitors can be tested (e.g., asdescribed in the examples).

The improved DNA polymerases of the present invention may be used forany purpose in which such enzyme activity is necessary or desired.Accordingly, in another aspect of the invention, methods ofpolynucleotide extension (e.g., PCR) using the polymerases are provided.Conditions suitable for polynucleotide extension are known in the art.(See, e.g., Sambrook et al., supra. See also Ausubel et al., ShortProtocols in Molecular Biology (4th ed., John Wiley & Sons 1999).Generally, a primer is annealed, i.e., hybridized, to a target nucleicacid to form a primer-template complex. The primer-template complex iscontacted with the DNA polymerase and nucleoside triphosphates in asuitable environment to permit the addition of one or more nucleotidesto the 3′ end of the primer, thereby producing an extended primercomplementary to the target nucleic acid. The primer can include, e.g.,one or more nucleotide analog(s). In addition, the nucleosidetriphosphates can be conventional nucleotides, unconventionalnucleotides (e.g., ribonucleotides or labeled nucleotides), or a mixturethereof. In some variations, the polynucleotide extension reactioncomprises amplification of a target nucleic acid. Conditions suitablefor nucleic acid amplification using a DNA polymerase and a primer pairare also known in the art (e.g., PCR amplification methods). (See, e.g.,Sambrook et al., supra; Ausubel et al., supra; PCR Applications:Protocols for Functional Genomics (Innis et al. eds., Academic Press1999). In other, non-mutually exclusive embodiments, the polynucleotideextension reaction comprises reverse transcription of an RNA template(e.g., RT-PCR). In some embodiments, the improved polymerases find usein 454 sequencing (Margulies, M et al. 2005, Nature, 437, 376-380).

Optionally, the primer extension reaction comprises an actual orpotential inhibitor of a reference or unmodified polymerase. Theinhibitor can inhibit, for example, the nucleic acid extension rateand/or the reverse transcription efficiency of a reference or unmodified(control) polymerase. In some embodiments, the inhibitor is hemoglobin,or a degradation product thereof. For example, in some embodiments, thehemoglobin degradation product is a heme breakdown product, such ashemin, hematoporphyrin, or bilirubin. In some embodiments, the inhibitoris an iron-chelator or a purple pigment. In other embodiments, theinhibitor is heparin. In certain embodiments, the inhibitor is anintercalating dye. In certain embodiments, the inhibitor is melanin,which has been described as a polymerase inhibitor. See, e.g, Ekhardt,et al., Biochem Biophys Res Commun. 271(3):726-30 (2000).

The DNA polymerases of the present invention can be used to extendtemplates in the presence of polynucleotide templates isolated fromsamples comprising polymerase inhibitors, e.g., such as blood. Forexample, the DNA polymerases of the present invention can be used toextend templates in the presence of hemoglobin, a major component ofblood, or in the presence of a hemoglobin degradation product.Hemoglobin can be degraded to various heme breakdown products, such ashemin, hematin, hematoporphyrin, and bilirubin. Thus, in certainembodiments, the DNA polymerases of the present invention can be used toextend templates in the presence of hemoglobin degradation products,including but not limited to, hemin, hematin, hematoporphyrin, andbilirubin. In certain embodiments, the hemoglobin degradation product ishemin. In some embodiments, the DNA polymerases of the present inventioncan be used to extend templates in the presence of about 0.5 to 20.0 μM,about 0.5 to 10.0 μM, about 0.5 to 5.0 μM, about 1.0 to 10.0 μM, about1.0 to 5.0 μM, about 2.0 to 5.0 μM, or about 2.0 to 3.0 μM hemin. Inother embodiments, the DNA polymerases of the present invention can beused to extend templates in the presence of at least about 0.5, 1.0,1.5, 2.0, 2.5, 3.0, 4.0, 5.0, 10.0, 20.0, or greater than 20 μM hemin.The breakdown products of hemoglobin include iron-chelators and purplepigments. Thus, in some embodiments, the DNA polymerases of the presentinvention can be used to extend templates in the presence ofiron-chelators and/or purple pigments. In other embodiments, the DNApolymerases of the present invention can be used to extend templates inthe presence of amounts of hemoglobin degradation products that wouldinhibit extension of the same template by a reference or control DNApolymerase.

The DNA polymerases of the present invention can be used to extendtemplates in the presence of heparin. Heparin is commonly present as ananticoagulant in samples isolated from blood. In some embodiments, theDNA polymerases of the present invention can be used to extend templatesin the presence of about 1.0 to 400 ng/μl, 1.0 to 300 ng/μl, 1.0 to 200ng/μl, 5.0 to 400 ng/μl, 5.0 to 300 ng/μl, 5.0 to 200 ng/μl, 10.0 to 400ng/μl, 10.0 to 300 ng/μl, or 10.0 to 200 ng/μl heparin. In someembodiments, the DNA polymerases of the present invention can be used toextend templates in the presence of at least about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 100, 150, 200, 250, 300,350, 400 ng/μl, or greater than 400 ng/μl of heparin. In otherembodiments, the DNA polymerases of the present invention can be used toextend templates in the presence of amounts of heparin that wouldinhibit extension of the same template by a reference or control DNApolymerase.

In some embodiments, an improved polymerase of the invention is used ina reverse transcription reaction. In some embodiments, the reversetranscription reaction is carried out in a mixture containing the RNAtemplate, one or more primer(s), and a thermostable DNA polymerase ofthe invention. The reaction mixture typically contains all four standarddeoxyribonucleoside triphosphates (dNTPs) and a buffer containing adivalent cation and a monovalent cation. Exemplary cations include,e.g., Mg²⁺, although other cations, such as Mn²⁺ or Co²⁺ can activateDNA polymerases. In other embodiments, the reverse transcriptionreaction is carried out with a thermo-active DNA polymerase of theinvention. In particular embodiments, the improved polymerase of theinvention allows for more efficient amplification of RNA templateswithout compromising the efficient amplification of a DNA template inthe presence of Mn²⁺ or Mg²⁺, as described in the examples.

In some embodiments, an improved polymerase of the invention increasesreverse transcription efficiency by reducing the reaction time requiredfor extending an RNA template. For example, an improved polymerasedescribed herein can significantly reduce the reaction time required totranscribe RNA to cDNA as compared to a control polymerase, therebyincreasing the reverse transcriptase efficiency. Without being limitedby theory, the improved polymerase can increase RT efficiency by, forexample, increasing the activity of the enzyme on an RNA template, suchas increasing the rate of nucleotide incorporation and/or increasing theprocessivity of the polymerase, thereby effectively shortening theextension time of an RNA template or population of RNA templates.Reaction times for the initial RT step are typically on the order of 30minutes or longer at 65 degrees C. when using an unmodified or controlpolymerase. Thus, in some embodiments, the improved polymerase cantranscribe an RNA template into cDNA in less than about 10 minutes, lessthan about 8 minutes, less than about 5 minutes, less than about 4minutes, less than about 3 minutes, or less than about 2 minutes at 65degrees C. In some embodiments, the improved polymerase can transcribean RNA template derived from Hepatitis C Virus (HCV) transcript JP2-5,containing the first 800 bases of HCV genotype Ib 5′NTR, into cDNA inless time or faster than a control polymerase. For example, the improvedpolymerase can transcribe 240 bases of the HCV JP2-5 RNA template intofull-length cDNA in about 15 seconds less, 30 seconds less, one minuteless, two minutes less, 3 minutes less, 4 minutes less, 5 minutes less,or about 10 minutes less than a control polymerase under identicalreaction conditions. In some embodiments, the improved polymerase cantranscribe 240 bases of the HCV JP2-5 RNA template into full-length cDNAfaster than a control polymerase, for example, about 5 seconds, 10seconds, 15 seconds, 30 seconds, 45 seconds, or 60 seconds or morefaster than a control polymerase under identical reaction conditions. Insome embodiments, the reaction conditions are those described in theExamples. In some embodiments, an improved polymerase described hereinis contacted with an RNA template at 65 degrees C. for 2 minutes in thereaction mixture described above. The extension step can be followed byPCR amplification of the extended template, as described in theexamples.

The most efficient RT activity in thermostable DNA polymerases has beenachieved using Mn²⁺ as the divalent metal ion activator. However, it iswell known that when Mn²⁺ is present in reactions the fidelity of DNApolymerases is lower. Unless one is trying to generate mutations, it isgenerally favored to maintain a higher fidelity. Fortunately, mostconventional sequencing, PCR and RT-PCR applications do not require highfidelity conditions because the detection systems generally are lookingat a population of products. With the advent of next generationsequencing, digital PCR, etc., the fidelity of the product is moreimportant and methods that allow for higher fidelity DNA synthesis arecritical. Achieving efficient RT activity using Mg²⁺ as the divalentmetal ion activator is an excellent way to substantially increase thefidelity of the DNA polymerase and allow for more reliable copying ofthe nucleic acid target. Accordingly, in some embodiments, the improvedpolymerase of the invention allows for efficient extension and/oramplification of RNA templates using Mg²⁺ as the divalent metal ionactivator, as described in the examples.

Because the polymerases described herein can also have increasedmismatch tolerance, the polymerases find use in methods where variationof the target template is likely and yet the template is neverthelessdesired to be amplified regardless of the variation at the targettemplate. An example of such templates can include, for example, viral,bacterial, or other pathogen sequences. In many embodiments, it isdesirable to determine simply whether an individual (human or non-humananimal) has a viral or other infection, regardless of the precise viralvariant that has infected the individual. As an example, one can use aprimer pair to amplify HCV using a polymerase of the invention anddetect the presence of the HCV even if the particular virus infectingthe individual has a mutation resulting in a mismatch at the primerhybridization site.

Target nucleic acids can come from a biological or synthetic source. Thetarget can be, for example, DNA or RNA. Generally, where amplicons aregenerated, the amplicons will be composed of DNA, though ribonucleotidesor synthetic nucleotides can also be incorporated into the amplicon.Where one wishes to detect an RNA, the amplification process willtypically involve the use of reverse transcription, including forexample, reverse transcription PCR (RT-PCR).

Specific target sequences can include, e.g., viral nucleic acids (e.g.,human immunodeficiency virus (HIV), hepatitis virus B (HBV),(cytomegalovirus (CMV), parvo B19 virus, Epstein-Barr virus, hepatitisvirus C(HCV), human papilloma virus (HPV), Japanese encephalitis virus(JEV), West Nile virus (WNV), St. Louis encephalitis virus (SLEV),Murray Valley encephalitis virus, and Kunjin virus), bacterial nucleicacids (e.g., S. aureus, Neisseria meningitidis, Plasmodium falciparum,Chlamydia muridarum, Chlamydia trachomatis), mycobacteria, fungalnucleic acids, or nucleic acids from animals or plants. In someembodiments, the target nucleic acids are animal (e.g., human) nucleicacids or are derived from an animal (e.g., human) sample (i.e., viral orother pathogenic organism nucleic acids may be present in a sample froman animal biopsy, blood sample, urine sample, fecal sample, saliva,etc.). In some embodiments, the target nucleic acids are, for example,human genetic regions that may include variants associated with disease(e.g., cancer, diabetes, etc.). Because in some embodiments thepolymerases of the invention have mismatch tolerance, such enzymes areparticularly useful, for example, where a diversity of related sequencescould be in a target sequence. As an example, the invention can be usedto detect viral pathogens, where the viral pathogens have sufficientvariation in their genomes to make it difficult or impossible to designa single or small set of primers that will amplify most or all possibleviral genomes or in cancer or other disease genetic markers wherevariation in sequence is known or likely to occur.

Other methods for detecting extension products or amplification productsusing the improved polymerases described herein include the use offluorescent double-stranded nucleotide binding dyes or fluorescentdouble-stranded nucleotide intercalating dyes. Examples of fluorescentdouble-stranded DNA binding dyes include SYBR-green (Molecular Probes).The double stranded DNA binding dyes can be used in conjunction withmelting curve analysis to measure primer extension products and/oramplification products. The melting curve analysis can be performed on areal-time PCR instrument, such as the ABI 5700/7000 (96 well format) orABI 7900 (384 well format) instrument with onboard software (SDS 2.1).Alternatively, the melting curve analysis can be performed as an endpoint analysis. Exemplary methods of melting point analysis aredescribed in U.S. Patent Publication No. 2006/0172324, the contents ofwhich are expressly incorporated by reference herein in its entirety.

In another aspect of the present invention, kits are provided for use inprimer extension methods described herein. In some embodiments, the kitis compartmentalized for ease of use and contains at least one containerproviding an improved DNA polymerase in accordance with the presentinvention. One or more additional containers providing additionalreagent(s) can also be included. In some embodiments, the kit can alsoinclude a blood collection tube, container, or unit that comprisesheparin or a salt thereof, or releases heparin into solution. The bloodcollection unit can be a heparinized tube. Such additional containerscan include any reagents or other elements recognized by the skilledartisan for use in primer extension procedures in accordance with themethods described above, including reagents for use in, e.g., nucleicacid amplification procedures (e.g., PCR, RT-PCR), DNA sequencingprocedures, or DNA labeling procedures. For example, in certainembodiments, the kit further includes a container providing a 5′ senseprimer hybridizable, under primer extension conditions, to apredetermined polynucleotide template, or a primer pair comprising the5′ sense primer and a corresponding 3′ antisense primer. In other,non-mutually exclusive variations, the kit includes one or morecontainers providing nucleoside triphosphates (conventional and/orunconventional). In specific embodiments, the kit includesalpha-phosphorothioate dNTPs, dUTP, dITP, and/or labeled dNTPs such as,e.g., fluorescein- or cyanin-dye family dNTPs. In still other,non-mutually exclusive embodiments, the kit includes one or morecontainers providing a buffer suitable for a primer extension reaction.

In another aspect of the present invention, reaction mixtures areprovided comprising the polymerases with increased reverse transcriptaseefficiency, mismatch tolerance, extension rate and/or tolerance of RTand polymerase inhibitors as described herein. The reaction mixtures canfurther comprise reagents for use in, e.g., nucleic acid amplificationprocedures (e.g., PCR, RT-PCR), DNA sequencing procedures, or DNAlabeling procedures. For example, in certain embodiments, the reactionmixtures comprise a buffer suitable for a primer extension reaction. Thereaction mixtures can also contain a template nucleic acid (DNA and/orRNA), one or more primer or probe polynucleotides, nucleosidetriphosphates (including, e.g., deoxyribonucleotides, ribonucleotides,labeled nucleotides, unconventional nucleotides), salts (e.g., Mn²⁺,Mg²⁺), labels (e.g., fluorophores). In some embodiments, the reactionmixtures contain a 5′-sense primer hybridizable, under primer extensionconditions, to a predetermined polynucleotide template, or a primer paircomprising the 5′-sense primer and a corresponding 3′ antisense primer.In some embodiments, the reaction mixtures containalpha-phosphorothioate dNTPs, dUTP, dITP, and/or labeled dNTPs such as,e.g., fluorescein- or cyanin-dye family dNTPs. In some embodiments, thereaction mixtures comprise an iron chelator or a purple dye. In certainembodiments, the reaction mixtures comprise hemoglobin, or a degradationproduct of hemoglobin. For example, in certain embodiments, thedegradation products of hemoglobin include heme breakdown products suchas hemin, hematin, hematophoryn, and bilirubin. In other embodiments,the reaction mixtures comprise heparin or a salt thereof. In certainembodiments, the reaction mixture contains a template nucleic acid thatis isolated from blood. In other embodiments, the template nucleic acidis RNA and the reaction mixture comprises heparin or a salt thereof.

In some embodiments, the reaction mixture comprises two or morepolymerases. For example, in some embodiments, the reaction mixturecomprises a first DNA polymerase having increased reverse transcriptaseefficiency compared to a control polymerase, and a second DNA polymerasehaving DNA-dependent polymerase activity. The second DNA polymerase canbe a wild-type or unmodified polymerase, or can be an improvedpolymerase having increased DNA-dependent polymerase activity. Suchreaction mixtures are useful for amplification of RNA templates (e.g.,RT-PCR) by providing both a polymerase having increased reversetranscriptase activity and a polymerare having DNA-dependent polymeraseactivity.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Library Generation

In brief, the steps in this screening process included librarygeneration, expression and partial purification of the mutant enzymes,screening of the enzymes for the desired properties, DNA sequencing,clonal purification, and further characterization of selected candidatemutants. Each of these steps is described further below.

Clonal Library Generation:

A nucleic acid encoding the polymerase domain of Z05 D580G_I709K DNApolymerase was subjected to error-prone (mutagenic) PCR between Blp Iand Bgl II restriction sites of a plasmid including this nucleic acidsequence. The primers used for this are given below:

Forward Primer: (SEQ ID NO: 30) 5′- CTACCTCCTGGACCCCTCCAA-3′; and,Reverse Primer: (SEQ ID NO: 31) 5′- ATAACCAACTGGTAGTGGCGTGTAA-3′PCR was performed using a Mg²⁺ concentration of 1.8 mM, in order togenerate a library with a desired mutation rate. Buffer conditions were50 mM Bicine pH 8.2, 115 mM KOAc, 8% w/v glycerol, and 0.2 mM eachdNTPs. A GeneAmp® AccuRT Hot Start PCR enzyme was used at 0.15 U/μL.Starting with 5×10⁵ copies of linearized Z05 D580G_I709K plasmid DNA perreaction volume of 50 μL, reactions were denatured using a temperatureof 94° C. for 60 seconds, then 30 cycles of amplification wereperformed, using a denaturation temperature of 94° C. for 15 seconds, anannealing temperature of 60° C. for 15 seconds, an extension temperatureof 72° C. for 120 seconds, and followed by a final extension at atemperature of 72° C. for 5 minutes.

The resulting amplicon was purified with a QIAquick PCR Purification Kit(Qiagen, Inc., Valencia, Calif., USA) and cut with Blp I and Bgl II, andthen re-purified with a QIAquick PCR Purification Kit. A Z05 D580G_I709Kvector plasmid was prepared by cutting with the same two restrictionenzymes and treating with alkaline phosphatase, recombinant (RAS,cat#03359123001) and purified with a QIAquick PCR Purification Kit. Thecut vector and the mutated insert were mixed at a 1:3 ratio and treatedwith T4 DNA ligase for 5 minutes at room temperature (NEB QuickLigation™ Kit). The ligations were purified with a QIAquick PCRPurification Kit and transformed into an E. coli host strain byelectroporation.

Aliquots of the expressed cultures were plated on ampicillin-selectivemedium in order to determine the number of unique transformants in eachtransformation. Transformations were pooled and stored at −70° C. to−80° C. in the presence of glycerol as a cryo-protectant.

The library was then spread on large format ampicillin-selective agarplates. Individual colonies were transferred to 384-well platescontaining 2× Luria broth with ampicillin and 10% w/v glycerol using anautomated colony picker (QPix2, Genetix Ltd). These plates wereincubated overnight at 30° C. to allow the cultures to grow and thenstored at −70° C. to −80° C. The glycerol added to the 2× Luria brothwas low enough to permit culture growth and yet high enough to providecryo-protection. Several thousand colonies were prepared in this way forlater use.

Extract Library Preparation Part 1—Fermentation: From the clonallibraries described above, a corresponding library of partially purifiedextracts suitable for screening purposes was prepared. The first step ofthis process was to make small-scale expression cultures of each clone.These cultures were grown in 96-well format; therefore there were 4expression culture plates for each 384-well library plate. 0.5 μL wastransferred from each well of the clonal library plate to a well of a 96well seed plate, containing 150 μL of Medium A (see Table 3 below). Thisseed plate was shaken overnight at 1150 rpm at 30° C., in an iEMS plateincubator/shaker (ThermoElectron). These seed cultures were then used toinoculate the same medium, this time inoculating 20 μL into 250 μLMedium A in large format 96 well plates (Nunc #267334). These plateswere incubated overnight at 37° C. with shaking. The expression plasmidcontained transcriptional control elements, which allow for expressionat 37° C. but not at 30° C. After overnight incubation, the culturesexpressed the clone protein at typically 1-10% of total cell protein.The cells from these cultures were harvested by centrifugation. Thesecells were either frozen (−20° C.) or processed immediately, asdescribed below.

TABLE 2 Medium A (Filter-sterilized prior to use) ComponentConcentration MgSO₄•7H₂O 0.2 g/L Citric acid•H₂O 2 g/L K₂HPO₄ 10 g/LNaNH₄PO₄•4H₂O 3.5 g/L MgSO₄ 2 mM Casamino acids 2.5 g/L Glucose 2 g/LThiamine•HCl 10 mg/L Ampicillin 100 mg/L

Extract Library Preparation Part 2—Extraction: Cell pellets from thefermentation step were resuspended in 25 μA Lysis buffer (Table 3 below)and transferred to 384-well thermocycler plates and sealed. Note thatthe buffer contained lysozyme to assist in cell lysis, and DNase toremove DNA from the extract. To lyse the cells the plates were incubatedat 37° C. for 15 minutes, frozen overnight at −20° C., and incubatedagain at 37° C. for 15 minutes. Ammonium sulfate was added (1.5 μL of a2M solution) and the plates incubated at 75° C. for 15 minutes in orderto precipitate and inactivate contaminating proteins, including theexogenously added nucleases. The plates were centrifuged at 3000×g for15 minutes at 4° C. and the supernatants transferred to a fresh 384-wellthermocycler plate. These extract plates were frozen at −20° C. forlater use in screens. Each well contained about 0.5-3 μM of the mutantlibrary polymerase enzyme.

TABLE 3 Lysis Buffer Concentration Component or Percentage Tris pH 7.550 mM EDTA 1 mM MgCl₂ 6 mM Tween 20 0.5% v/v Lysozyme (from powder) 1mg/mL DNase I 0.05 Units/μL

Example 2 Identification of Mutant DNA Polymerases with Improved ReverseTranscription Efficiency

Screening Crude Protein Extract Libraries for Improved ReverseTranscription Efficiency: The extract library was screened by comparingCp (Crossing Point) values from growth curves generated by fluorescent5′ nuclease (TaqMan) activity of crude enzyme extracts in a RT-PCRsystem from amplification of a 240 base pair amplicon from Hepatitis CVirus (HCV) transcript JP2-5, containing the first 800 bases of HCVgenotype Ib 5′NTR in pSP64 poly(A) (Promega).

Reactions were carried out on the Roche LC 480 kinetic thermocycler in384 well format with each well containing 3 μL of an individual enzymeextract diluted 10-fold with buffer containing 20 mM Tris-HCl, pH 8, 100mM KCl, 0.1 mM EDTA, and 0.1% Tween-20 added to 12 μL of RT-PCR mastermix described in Table 4. The thermocycling conditions were: 2 minute at50° C. (“UNG” step); 2 minute at 65° C. (“RT” step); 5 cycles of 94° C.for 15 seconds followed by 62° C. for 30 seconds; and 45 cycles of 91°C. for 15 seconds followed by 62° C. for 30 seconds.

TABLE 4 Component Concentration Tricine pH 8.3 50 mM KOAc 60 mM Glycerol5% (v/v) DMSO 2% (v/v) Primer 1 200 nM Primer 2 200 nM TaqMan Probe 100nM Aptamer 200 nM dATP 200 μM dCTP 200 μM dGTP 200 μM dUTP 400 μM UNG .2Units/μL RNA Target 6666 copies/μL Mg(OAc)₂ 2 mM

Approximately 5000 clones were screened using the above protocol. Fortyclones were chosen from the original pool for rescreening based onearliest Crossing Point (Cp) values and fluorescent plateau values abovean arbitrary cut off as calculated by the Abs Quant/2^(nd) derivativemax method. Culture wells corresponding to the top extracts were sampledto fresh growth medium and re-grown to produce new culture platescontaining the best mutants, as well as a number of parental Z05D580G_I709K cultures to be used for comparison controls. These cultureplates were then used to make fresh crude extracts which were rescreenedwith the same RNA target and conditions as previously described for theoriginal screen. Table 5 shows average Cp values obtained from thefluorescent signal increase due to 5′ hydrolysis of a FAM labeled probe.Results show that clone 0691-L24 amplifies the RNA target with higherefficiency than the Z05_D580G_I709K parental.

TABLE 5 Clone Average Cp 0691-L24 19.1 Z05 D580G_I709K 28.0

The DNA sequence of the mutated region of the polymerase gene wassequenced to determine the mutation(s) that were present in any singleclone. Clone 0691-L24 was chosen for further testing, so mutantpolymerase protein was expressed in flask culture, purified tohomogeneity, and quantified.

Use of Fully Purified 0691-L24 Mutant in Mg²⁺-Based RT-PCR: Purified andquantified mutant 0691-L24 was compared to parental Z05_D580G_I709K inTaqMan Mg²⁺-based RT-PCR. Reverse transcription and PCR efficiencieswere measured by comparing Cp values from amplifications of JP2-5 RNAtranscript and pJP2-5 DNA linear plasmid digested with the restrictionendonuclease EcoRI. Oligonucleotides and Master Mix conditions (Table 4)were the same as used in the original screen. Each reaction had either100,000 copies of JP2-5 transcript RNA, 100,000 copies of pJP2-5 linearplasmid DNA, or 1000 copies of pJP2-5 linear plasmid DNA. All targetswere amplified with Primer 1 and Primer 2, as described above, induplicate reactions to generate a 240 base pair amplicon. All reactionswere performed on the Roche Light Cycler 480 thermal cycler with areaction volume of 15 μL. Crossing Point (Cps) were calculated by theAbs Quant/2^(nd) derivative max method and averaged. Master Mixconditions were the same as those described previously in Table 4 exceptreactions were carried out using a range of DNA Polymeraseconcentrations from 5 nM-40 nM. The thermocycling conditions were: 2minute at 50° C. (“UNG” step); 2 minute at 65° C. (“RT” step); 5 cyclesof 94° C. for 15 seconds followed by 62° C. for 30 seconds; and 45cycles of 91° C. for 15 seconds followed by 62° C. for 30 seconds. Table6 shows Cp values obtained from fluorescent signal increase due tocleavage of the TaqMan probe at 20 nM enzyme condition.

TABLE 6 RNA 10⁵ DNA 10⁵ DNA 10³ Enzyme copies Cp copies Cp copies Cp Z05D580G_I709K 28.8 17.5 24.4 0691-L24 18.9 17.3 24.0

The results indicate that mutant DNA polymerase 0691-L24 allows for moreefficient amplification of RNA target without compromise of PCRefficiency on DNA target, as compared to parental Z05 D580G_I709K.

Determination of Phenotype-Conferring Mutation(s): Sequencing resultsrevealed that the polymerase expressed by clone 0691-L24 carriesmutations N629D and I640F in addition to the parental D580G and I709Kmutations. A Z05 D580G_I709K_I640F mutant was constructed by subcloning,purified, quantified, and compared to 0691-L24 (Z05D580G_I709K_N629D_I640F) and parental Z05 D580G_I709K in Mg²⁺ activatedTaqMan RT-PCR with varying KOAc concentration from 40 mM-110 mM and 25nM purified enzyme. Master Mix conditions were the same as thosedescribed previously in Table 4. Each reaction had either 100,000 copiesof JP2-5 RNA transcript, 100,000 copies of pJP2-5 linear plasmid DNA, or1000 copies of pJP2-5 linear plasmid DNA. All targets were amplifiedwith the same primer set in duplicate reactions to generate a 240 basepair amplicon. The PCR and RT-PCR efficiencies were determined bycomparing Cp values between DNA and RNA. All reactions were performed onthe Roche Light Cycler 480 thermal cycler with a reaction volume of 15μL. Crossing Point (Cps) were calculated by the Abs Quant/2^(nd)derivative max method and Cps were averaged. The thermocyclingconditions were: 2 minutes at 50° C. (“UNG” step); 65° C. for 2 minutes(“RT” step); 5 cycles of 94° C. for 15 seconds followed by 62° C. for 30seconds; and 45 cycles of 91° C. for 15 seconds followed by 62° C. for30 seconds. Table 8 shows the Cp values obtained from fluorescent signalincrease due to cleavage of the TaqMan probe at the 60 mM KOAccondition.

TABLE 7 RNA 10⁵ DNA 10⁵ DNA 10³ Enzyme copies Cp copies Cp copies Cp Z05D580G_I709K 28.1 17.2 24.1 0691-L24 18.8 17.2 24.3 Z05 D580G_I709K_I640F19.6 17.0 24.2

0691-L24 (Z05 D580G_I709K_N629D_I640F) and Z05 D580G_I709K_I640F havesimilar Cp values on both RNA and DNA targets, demonstrating that theI640F mutation confers the observed improvement in RT-PCR performance.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, sequence accessionnumbers, patents, and patent applications cited herein are herebyincorporated by reference in their entirety for all purposes.

What is claimed is:
 1. A DNA polymerase having increased reversetranscriptase efficiency compared with a control DNA polymerase, thephrase-wherein the DNA polymerase comprises an amino acid sequence atleast 90% identical to SEQ ID NO:1 wherein the amino acid of the DNApolymerase corresponding to position 640 of SEQ ID NO:1 is any aminoacid other than I, and wherein the control DNA polymerase has the sameamino acid sequence as the DNA polymerase except that the amino acid ofthe control DNA polymerase corresponding to position 640 of SEQ ID NO:1is I.
 2. The DNA polymerase of claim 1, wherein the DNA polymerasecomprises an amino acid sequence at least 95% identical to SEQ ID NO:1.3. The DNA polymerase of claim 1, wherein the amino acid of the DNApolymerase corresponding to position 640 of SEQ ID NO:1 is F.
 4. The DNApolymerase of claim 1, wherein the amino acid corresponding to position580 of SEQ ID NO:1 is any amino acid other than D.
 5. The DNA polymeraseof claim 1, wherein the amino acid corresponding to position 580 of SEQID NO:1 is selected from the group consisting of L, G, T, Q, A, S, N, R,and K.
 6. The DNA polymerase of claim 1, wherein the amino acidcorresponding to position 709 of SEQ ID NO:1 is any amino acid otherthan I.
 7. The DNA polymerase of claim 1, wherein the amino acidcorresponding to position 709 of SEQ ID NO:1 is selected from the groupconsisting of K, R, S, G, and A.
 8. The DNA polymerase of claim 1,wherein the amino acid corresponding to position 580 of SEQ ID NO:1 isany amino acid other than D; and wherein the amino acid corresponding toposition 709 of SEQ ID NO:1 is any amino acid other than I.
 9. The DNApolymerase of claim 8, wherein the amino acid corresponding to position580 of SEQ ID NO:1 is selected from the group consisting of L, G, T, Q,A, S, N, R, and K; and wherein the amino acid corresponding to position709 of SEQ ID NO:1 is selected from the group consisting of K, R, S, G,and A.
 10. The DNA polymerase of claim 9, wherein the amino acidcorresponding to position 580 of SEQ ID NO:1 is G; and wherein the aminoacid corresponding to position 709 of SEQ ID NO:1 is K.
 11. The DNApolymerase of claim 1, wherein the amino acid corresponding to position640 of SEQ ID NO:1 is F, the amino acid corresponding to position 709 ofSEQ ID NO:1 is K, and the amino acid corresponding to position 580 ofSEQ ID NO:1 is G.
 12. A recombinant nucleic acid encoding the DNApolymerase according to claim
 1. 13. A method for conducting primerextension, comprising: contacting a DNA polymerase as in claim 1 with aprimer, a polynucleotide template, and nucleoside triphosphates underconditions suitable for extension of the primer, thereby producing anextended primer.
 14. The method of claim 13, wherein the template isRNA.
 15. The method of claim 13, wherein the primer extension methodcomprises a polymerase chain reaction (PCR).
 16. A kit for producing anextended primer, comprising: at least one container providing a DNApolymerase as in claim
 1. 17. The kit according to claim 16, furthercomprising one or more additional containers selected from the groupconsisting of: (a) a container providing a primer hybridizable, underprimer extension conditions, to a predetermined polynucleotide template;(b) a container providing nucleoside triphosphates; and (c) a containerproviding a buffer suitable for primer extension.
 18. A reaction mixturecomprising a DNA polymerase as in claim 1, at least one primer, apolynucleotide template, and nucleoside triphosphates.
 19. The reactionmixture of claim 18, wherein the polynucleotide template is RNA.
 20. Thereaction mixture of claim 18, further comprising a second thermostableDNA polymerase.